Tumor-selective combination therapy

ABSTRACT

The therapies described herein can be selectively lethal toward a variety of different cancer cell types and cancer conditions in a subject. The combination therapies described herein can be useful for the management, treatment, control, or adjunct treatment of diseases, where the selective legality is beneficial in chemotherapeutic therapy, particularly where the disease is accompanied by elevated levels of NQO1.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/783,344, filed Oct. 8, 2015, which is a national phase applicationunder 35 U.S.C. § 371 of International Patent Application No.PCT/US2014/033400, filed Apr. 8, 2014, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Patent Application No. 61/810,008,filed Apr. 9, 2013, each of which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under contract numberCA102792 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

A fundamental challenge in cancer treatment is the discovery ofcompounds that are toxic to cancer cells but not healthy cells. Asalient feature of cancer is rapid and unrestricted cell division. Thevast majority of traditional chemotherapeutics target rapidly dividingcells by disrupting the cell cycle, causing cell death. Because somehealthy tissues require cell division as part of their function,antiproliferative cytotoxins can also kill healthy cells, resulting insevere, dose-limiting side effects. Accordingly, new therapeutic methodsand new cellular targets must be identified that better differentiatehealthy and cancerous cells. The targets may be present in only a smallfraction of cancer patients, thereby allowing for a personalizedstrategy to treat cancer.

Quinone-containing molecules are frequently cytotoxic and harm cellsthrough one of two mechanisms. Many quinones are conjugate additionacceptors and readily alkylate nucleophilic species such as DNA andcysteine residues. Quinones are also substrates for 1-electronreductases, such as cytochrome P450s, cytochrome b5, xanthine oxidase,and glutathione reductase. Reduction of quinones by these enzymesgenerates a highly reactive semiquinone that can damage biomoleculesdirectly, or can be oxidized by dissolved oxygen resulting in theformation of an equivalent of superoxide anion radical and the parentquinone. Thus, 1-electron reduction of quinones can catalytically createreactive oxygen species (ROS) that damage the cell.

NAD(P)H quinone oxidoreductase (NQO1, DT diaphorase) is an FAD-dependent2-electron reductase whose primary function is to protect the cell fromcytotoxins, especially quinones. Although generally identified as acytosolic protein, NQO1 has been identified in subcellular compartmentssuch as the mitochondria and nucleus. By reducing quinones in a2-electron process, NQO1 bypasses the toxic semiquinone and formshydroquinones, which are commonly unreactive toward oxygen.Hydroquinones are then conjugated with molecules such as glutathione,glucose, or sulfate, and excreted by the cell. However, somehydroquinone-containing molecules are unstable and react with oxygen intwo 1-electron oxidations back to the quinone, generating ROS. Therelative stability of hydroquinones toward air oxidation cannot bepredicted based on molecular structure and it does not correlate withreduction potential.

NQO1 has attracted much attention as a potential target for thetreatment of cancer because it has been shown to be frequently expressedat much higher levels in tumors relative to adjacent healthy tissue,particularly in the case of lung cancer. In addition, NQO1 activityappears to increase during tumor progression. Other than for lung,breast, and colon tissues, relatively little data on the levels of NQO1in normal tissues have been reported. Whereas low levels of NQO1 arereported in bone marrow and liver cells—two tissues frequently damagedby chemotherapeutics—relatively high levels of NQO1 have been noted instomach and kidney cells.

The prospect of discovering toxins that are activated, instead ofdeactivated, by NQO1 has attracted researchers for many years. Suchmolecules would turn this normally cytoprotective enzyme into aliability for the cell. Two general classes of molecules have beendiscovered that fit this description: DNA alkylators whoseelectrophilicity is increased after bioreduction, and redox cyclingmolecules that generate ROS catalytically after reduction. Examples ofsuch DNA alkylators include Mitomycin C, EO9, and MeDZQ, and examples ofsuch ROS generators include β-lapachone (β-lap) and streptonigrin, thecytotoxic mechanisms of which each involve NQO1-mediated bioreduction.These classes of molecules are composed almost exclusively ofquinone-containing compounds.

The concentration of β-lap delivered to cells may induce different formsof cell death, with lower concentrations inducing apoptosis and higherconcentrations initiating calcium-dependent necroptosis. In addition toROS generation in RBCs, the poor aqueous solubility of β-lapnecessitates the use of hydroxypropyl-β-cyclodextrin (HPβCD) as asolubility aid, high concentrations of which cause hemolysis of RBCs invitro. To address the issues of compound instability and damage to RBCs,the Boothman and Gao groups have designed a micellar formulation ofβ-lap that demonstrates greatly improved PK properties and efficacy inmurine tumor models (Blanco, Boothman, Gao et al., Cancer Res. 2010, 70,3896).

While personalized medicine strategies have produced life-savinganticancer drugs, they affect only a small percentage of cancerpatients. Because NQO1 levels are highly elevated in a large number ofsolid tumors, a treatment that successfully exploits NQO1 levels couldbenefit a significant fraction of all cancer patients. Thus, newtherapeutic methods that exploit elevated NQO1 levels and that canselectively inhibit or kill cancer cells are needed to benefit largernumbers of cancer patients.

SUMMARY

The invention provides compounds, compositions and methods to treatcancer and cancer tumor cells, for example, tumor cells having elevatedlevels of NQO1. The invention also provides novel combination therapyincluding the administration of NQO1 bioactivatable drugs in combinationwith DNA repair inhibitors to provide a tumor-selective therapy. In oneembodiment, inhibiting base excision repair synergistically enhancesbeta-lapachone-mediated cell death for tumor-selective therapy ofcancers such as pancreatic cancer.

NQO1 bioactivatable drugs include DNQ analogs and prodrugs that are NQO1substrates, and ß-lapachone and its prodrugs or analogues. These drugsmake DNA repair inhibitors tumor-selective. Such DNA repair inhibitorscan include inhibitors of DNA base excision (BE), single strand break(SSB), and double strand break (DSB) repair.

1. NQO1 bioactivatable drugs can be used in a tumor-selective manneragainst cancers with defects in base excision repair (BER). Dataindicate that BER processes are deficient (e.g., XRCC1) in specificcancers known to have elevated levels of NQO1 (i.e., breast cancers).For example, XRCC1 knockdown, whose levels have been shown to beselectively deficient in breast cancer, shows enhanced lethality inresponse to NQO1 bioactivatable drugs. XRCC1 knockdown and other BERdeficiencies can result in increased DNA single strand breaks and APsites. Prolonged AP site, SSBs or DNA double strand breaks (DSBs) causedin a tumor-selective manner by NQO1 bioactivatable drug treatment,results in tumor-specific lethality and synergy, with dramatictumor-selective, NQO1-dependent metabolic changes. Defects in Ogg1 arean exception because its knockdown made cells resistant to NQO1bioactivatable drugs. These data highlight that the initial DNA lesionsare 8-oxoguanine base lesions, an unreported finding at the heart ofinhibiting BER processes. The formation of dramatic levels of8-oxoguanine (8-OG) in pancreatic cancer cells in an NQO1-dependentmanner has also been shown.

2. NQO1 bioactivatable drugs can be used in a tumor-selective manner incombination with poly(ADP-ribosyl) polymerase I (PARP1) inhibitors, suchas the PARP1 inhibitors described herein. PARP1 inhibitors lacktumor-selectivity and NQO1 bioactivatable drugs afford this selectivity.All current published data regarding NQO1 bioactivatable drugs assertthat PARP1 hyperactivation is required for tumor-selective lethality;therefore inhibiting PARP1 would not be indicated. This is not the casein long-term survival responses, because inhibiting PARP1 preventsrepair of SSBs and/or DSBs. Cells die by a different, classicalapoptotic mechanism, versus by programmed necrosis caused by NQO1bioactivatable drugs alone.

All known PARP1 inhibitors tested show synergy with NQO1 bioactivatabledrugs in various cancers that over-express NQO1. Cells die by activatingcaspases and do not undergo programmed necrosis. Nevertheless,tumor-selective lethality is observed in each case. Data obtainedinclude that nontoxic doses of PARP1 inhibitors synergize with nontoxicdoses of NQO1 bioactivatable drugs. Data with ß-lap and DNQ analogsstrongly support this observation. Additionally, dicoumarol prevents thesynergy between NQO1 bioactivatable drugs and PARP1 inhibitors, and thesynergy is not noted in NQO1-deficient cells since these cells lack DNAdamage caused by the lack of NQO1-mediated futile redox cycle.

3. AP-site modifying drugs (e.g., methoxyamine, MeOX) can be used incombination with NQO1 bioactivatable drugs for NQO1-dependent,tumor-selective lethality. NQO1 bioactivatable drugs cause predominantlyDNA base damage (e.g., 8-oxyguanine (8-OG)), and loss of Ogg1 (aglycosylase that detects 8-oxoguanine) results in resistance to thesedrugs. When 8-oxoguanine is repaired, massive levels of AP sites areformed. While PARP1 can bind AP sites, it was not known that PARP1 couldbind AP sites modified by methoxyamine or other AP-modifying drugs. Wedemonstrated that MeOX enhances NQO1 bioactivatable drugs, and viceversa that NQO1 bioactivatable drugs make MeOX tumor-selective. Dataobtained include that nontoxic doses of MeOX enhance nontoxic doses ofNQO1 bioactivatable drugs, and that MeOX enhances loss of ATP, and moreimportantly significantly prevents ATP recovery after toxic or nontoxicdoses of NQO1 bioactivatable drugs.

For all three of the methods described above and herein below, muchlower doses of NQO1 bioactivatable drugs can be used in all threeversions of the combination therapies, avoiding dose-related toxicity(i.e., methemoglobinemia) with ß-lapachone, its prodrugs, and analogues,and especially DNQ analogues.

The invention further provides methods for predicting and determiningthe efficacy of NQO1 bioactivatable drugs and/or their combination withbase excision repair (BER) enzyme inhibitors, PARP1 inhibitors, AP basemodifying drugs, or a combination thereof. The methods can includeanalyzing and monitoring NQO1:catalase ratios in cells or in a subjectto determine if the cells or patient, or tissues of the subject, have arelatively high, medium, or low NQO1:catalase ratio, thereby allowingfor a determination of the likely efficacy of the actives or combinationof actives.

Another aspect of the invention provides pharmaceutical compositionsthat contain at least one β-lapachone or derivative thereof, or a DNQcompound, for example, a compound of Formula (I) and a pharmaceuticallyacceptable diluent, carrier, or excipient, optionally in combinationwith a base excision repair (BER) enzyme inhibitor, a PARP1 inhibitor,or an AP base modifying drug (e.g., MeOX). The invention also providesfor the use of these compounds and combinations thereof for thepreparation of pharmaceutical compositions, and the subsequent use ofthe compositions in the treatment of patients or subjects. Patients orsubjects can be mammals, including humans.

A further aspect of the invention provides methods of treating, killing,or inhibiting the growth of tumor cells that have elevated NQO1 levelsor a tumor having cells that have elevated NQO1 levels, where at leastone tumor cell is exposed to a therapeutically effective amount of acompound, a pharmaceutically acceptable salt or solvate thereof, acombination of compounds, or a pharmaceutically acceptable compositionthereof. The administration of the compounds to cells, or to a patientin need of therapy (including vulnerable tumors known to be deficient inDNA base excision, single strand, or DNA double strand break repair),can be concurrent or sequential, with a quinone compound administered atleast concurrently or after a base excision repair (BER) enzymeinhibitor, a PARP1 inhibitor, or an AP base modifying drug.

Thus, the invention provides the use of a NQO1 bioactivatable drug incombination with a base excision repair (BER) enzyme inhibitor or otheractive agent described herein for killing or inhibiting the growth ofcancer cells in vitro, or cancer cells in a patient that has cancerouscells or a cancer tumor. The invention further provides the use of aNQO1 bioactivatable drug in combination with a base excision repair(BER) enzyme inhibitor or other active agent described herein for themanufacture of a medicament for killing or inhibiting the growth ofcancer cells in vitro, or cancer cells in a patient that has cancerouscells or a cancer tumor, wherein the medicament comprises an effectivelethal or inhibitory amount of the NQO1 bioactivatable drug and the baseexcision repair (BER) enzyme inhibitor or other active agent describedherein. In other embodiments, one medicament can include a NQO1bioactivatable drug and a second medicament can include a base excisionrepair (BER) enzyme inhibitor or other active agent described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention. However, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIGS. 1A-H. Expression of NQO1 and Catalase in pancreatic tumor vs.associated normal tissue. (A, B) Matched tumor and associated normaltissue from 59 pancreatic patients were analyzed for NQO1 (A) andCatalase (B) levels, showing significant Catalase over-expression innormal vs. tumor tissue. (C, F) NQO1 levels from 349 patient samples and85 pancreatic cancer cell lines. (D, G) Catalase levels monitored as in(C, F). (E, H) NQO1/catalase ratios can be a major determinant in theefficacy of NQO1-bioactivatable drugs. For (C-H), n=232 patient samples.

FIGS. 2A-B. NQO1:Catalase ratios in pancreatic cancer vs. normal tissueare a major determinant of efficacy for NQO1 bioactivatable drugs, ß-lapand DNQ. (A) Pancreatic cancer cells express elevated NQO1, but lowerCatalase levels. Metabolism of ß-lap results in extensive H₂O₂production that easily overwhelms low tumor Catalase levels. (B) Incontrast, normal pancreatic tissue expresses low NQO1 and abundantCatalase levels and are, therefore, protected from NQO1 bioactivatabledrugs. Patient sample enzyme assays have validated these NQO1:Catalaseratios.

FIGS. 3A-F. Functional inhibition of NQO1 by shRNA-NQO1 knockdownprotects from cell death after β-lap exposures. (A) Mechanism of β-lapredox cycling; DNQ and its derivatives can follow an analogousmechanism. (B) NQO1 protein levels and enzyme activities in Mia Paca-2parental, knockdown (17-1, 17-3, and 17-7), and Non-Silencing (NS, MiaPaca-2 shSCR) clones. (C) Long-term relative survival assays of MiaPaca-2 parental and NQO1 knockdown cells with β-lap or β-lap plusdicumoral (DIC) for 2 hour exposures at the indicated doses. (D)Survival of NQO1 knockdown clones (17-1, 17-3, 17-7) exposed to β-lap (6μM, 2 h) was significantly rescued by low doses of DIC (μM, 2 h)compared to doses required to rescue Mia Paca-2 NS clones (***p<0.001).(E) β-Lap dosage for LD₅₀ of Mia Paca-2 knockdown clones and differentpancreatic cancer cell lines (BXPC3, Capan1, ASPC1, HS766T, Capan2, andCFPAC1) (R²=0.9053). (F) β-Lap lethality in various pancreatic cancercells, with or without dicoumarol (DIC, 50 μM) or BAPTA-AM (5 μM, 1 h)treatments.

FIGS. 4A-C. β-Lap-induced DNA damage is significantly decreased by NQO1shRNA-knockdown. (A) NS and shRNA-NQO1 knockdown Mia Paca-2 clones(17-1, 17-3, 17-7 and NS) were exposed to β-lap, +50 μM DIC and assessedfor DNA damage using comet assays. Cells were also exposed to 2 mM H₂O₂for 2 hours as positive controls. (B) Comet tail lengths were measuredusing Image J software (a.u., arbitrary unit) (***, p<0.001). (C) ß-Laptreatment causes extensive 8-oxoguanine (8-OG) base damage in Mia Paca-2pancreatic cancer cells. Thus, ß-lap causes SSBs and extensive basedamage.

FIGS. 5A-F. Metabolic changes after β-lap or DNQ treatments. Mia Paca-2cells were treated with β-lap (6 μM) (A), DNQ or a DNQ derivative(DNQ87) (C, D)+/−DIC (50 μM) and survival was monitored. Mia PaCa-2cells exposed to ß-lap+/−DIC were also assessed for changes in ATP (B),glucose consumption (E) and lactate production in the media (F) at theindicated times.

FIGS. 6A-B. siRNA-mediated knockdown of the Ogg1 glycosylase rendersß-lap-treated pancreatic cancer cells resistant to ß-lapachone. Twoseparate experiments are shown. Mia-Paca2 pancreatic cancer cells wereexposed to siRNA-scrambled (NS) or siRNA-specific for Ogg1 (siOGG1) for24 hours, then cells were treated with ß-lapachone for 2 hours at theindicated doses. Survival, measured by colony forming ability assayswere then performed and graphed with ß-lapachone doses used, asillustrated in (A) and (B).

FIG. 7. NQO1 bioactivatable drugs cause SSBs and base lesions in atumor-specific manner, thus they can be used to make DNA repairinhibitors tumor-selective.

FIGS. 8A-H. PARP1 inhibitors synergize with NQO1 bioactivatable drugs.(A-D) Mia Paca-2 cells were pre-incubated with various PARP1 inhibitors(10 μM) then with deoxynyboquinone (DNQ), as indicated, for 2 hours withinhibitors. (E-H) AG014699 enhances ß-lap. Relative survival was thenassessed using a 7-day DNA assay (Huang et al., Cancer Res., 2012,72(12), 3038-3047) (***, p<0.001).

FIGS. 9A-F. Methoxyamine (MeOX) enhances β-lap-induced NQO1-drivenlethality. Mia Paca-2 cells were treated with indicated doses ofβ-lap+/−MeOX (12 mM), or β-lap alone for 2 hours. Endpoints examined:(A) Colony forming assays show synergy of MeOX+ß-lap; (B) MeOX treatmentmodifies AP sites induced by β-lap in Mia Paca-2 cells; (C) MeOXenhances β-lap-induced ATP loss; (D) MeOX blocks ATP recovery aftersublethal β-lap doses; (E) β-lap+MeOX co-treatment enhances γH2AX fociformation. MeOX+a sublethal β-lap dose (2 μM) was equivalent to a lethalβ-lap dose (6 μM); and (F) MeOX (6 mM)+β-lap (2 μM) inducedsignificantly greater apoptosis (TUNEL+ cells) in an NQO1-dependentmanner than β-lap (4 μM) alone. Dicoumarol (DIC, 50 μM, 2 h) blockedsynergy.

FIGS. 10A-D. β-Lapachone has significant antitumor efficacy against MIAPaCa-2 tumor xenografts. (A) Body weight changes of mice bearingorthotopic MIA PaCa-2 tumors. (B) Kaplan-Meier survival for pancreaticantitumor efficacy experiments described in (A). Log-rank analyses wereperformed comparing survival curves (***p<0.0001) for HPβ-CD vs.β-lap-HPβ-CD at 20 or 30 mg/kg, iv. Results are combined survival datafrom three similar experiments. (C) Bioluminescent images (BLI) of micebearing spleen-implanted pancreas cancers before and after treatmentwith HPβ-CD or β-lap-HPβ-CD (Arq761) at 20 or 30 mg/kg, iv. (D)Quantification of pancreatic tumor burden. BLI (photons per second) weredetermined before and 12 days post-therapy with HPβ-CD or β-lap-HPβ-CDat 20 or 30 mg/kg, iv. Results are means, +SE (n=5). Student's t tests(***p<0.005) were performed comparing HPβ-CD vs. β-lap-HPβ-CD at 20 or30 mg/kg, iv. V, Vehicle (HPβ-CD).

FIG. 11. Formulas of certain DNQ compounds, according to variousembodiments of the invention. When n=1-30, n can be specifically anyinteger from 1 to 30, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

FIG. 12. Examples of specific DNQ compounds, according to variousembodiments.

FIGS. 13A-F. DNQ87 (IB-DNQ) works at much lower doses versus ß-lapachoneand at doses equivalent to the parental DNQ compound. It is effectiveagainst breast cancer cells in an NQO1-dependent manner, as well astriple-negative breast cancer cells (A-F, for various cell types).

FIGS. 14A-H. Efficacy of DNQ87 increases in an NQO1-dependent manner(IB-DNQ exposure at various concentrations, A-H).

FIGS. 15A-D. IB-DNQ Cytotoxicity: “Noncaspase-mediated Cell Death”.DNQ87 causes cell death that can be blocked by dicoumarol, catalase, andBAPTA-AM (a calcium chelator) (A), in descending order and consistentwith the proposed pathway of cell death caused by NQO1 bioactivatabledrugs (B). (C) PARP1 hyperactivation caused by DNQ87 exposure measuredby PAR-PARP1 formation, highlighted by μ-calpain-mediated p53 cleavage(C) and atypical cleavage of PARP1 to ^(˜)60 kDa proteolytic fragmentsduring cell death (D).

FIGS. 16A-F. DNQ87 causes DNA lesions (DNA double strand breaks) in adelayed manner, monitored by gamma-H2AX, phosphorylation of ATM atser1981, and phosphorylation of DNA-PKcs at site Thr1892 (A-F).

FIGS. 17A-B. SSB-PARP1-DSB Temporal Sequence. MCF-7 breast cancer cellswere exposed to DNQ87 (IB-DNQ), with or without dicoumarol (DIC, 50 μM)and survival (A) or temporal sequence of PAR-PARP1 (PAR) formation, aswell as phosphorylations if H2AX (g-H2AX), ATM were monitored atspecific sites indicated and monitored using alpha-tubulin levels forloading (B).

FIG. 18. The therapeutic window of DNQ87 using dicoumarol to mimic lowlevels of NQO1 in normal tissue compared to using dicoumarol to mimicnormal tissue in NQO1 overexpressing MCF-7 human breast cancer cells (attime points 30 minutes, 60 minutes, 2 hours, 24 hours, 48 hours, and 72hours).

FIGS. 19A-C. Exposure to NQO1 bioactivatable drugs causes extensive8-oxoguanine levels. A) ß-lapachone exposure causes extensive formationof 8-oxoguanine. B, C) Two separate experiments are shown. Mia-Paca2pancreatic cancer cells were exposed to siRNA-scrambled (NS) orsiRNA-specific for Ogg1 (siOGG1) for 24 hours, then cells were treatedwith ß-lapachone for 2 hours at the indicated doses. Survival, measuredby colony forming ability assays were then performed and graphed withß-lapachone doses used.

FIGS. 20A-C. Determination of lethal vs. sublethal doses of methoxyamine(MeOX or MX, as indicated); A, MeOX with β-lapachone; B, MeOX; C, MeOX.

FIGS. 21A-D. Inhibition of BER mediated by Methoxyamine (MeOX) (A, B) orby XRCC1 shRNA knockdown (C) enhances the lethal effects of β-lap asmeasured using colony forming ability assays; (D), surviving fractionshown.

FIGS. 22A-B. Combined treatment of BER inhibitors and β-lap increasesDNA damage (A) and cell death monitored by TUNEL assays (B), consistentwith programmed necrosis.

FIGS. 23A-D. Inhibition of BER pathway enhances β-lap induced ATP lossand represses its recovery (A-D).

FIGS. 24A-D. MeOX suppresses ATP loss recovery after lethal (6 μM (A) or4 μM (B)) or sublethal (3 μM (C) or 2.5 μM (D)) β-lap treatments 0.

FIGS. 25A-B. Methyl pyruvate (MP) suppresses β-lapachone-induced celldeath; (A) relative ATP level; (B) relative cell survival.

FIGS. 26A-C. MeOX attenuates methyl pyruvate effect in protecting cells;(A) β-lap control and addition of MP; (B) β-lap and MeOX, and (C) β-lapand MP or β-lap and MeOX.

FIGS. 27A-B. Methoxyamine (MeOX) accelerates PARP1hyperactivation-induced NAD+/ATP loss, further accelerating O₂consumption by an NQO1-dependent process in ß-lapachone-exposed MiaPaCa-2 pancreatic cells. A. ß-Lapachone enhances oxygen consumptionrates (OCR) in Mia PaCa2 cells. Lethal doses of ß-lap cause dramatic OCRspikes, and subsequent losses of all metabolic capacity. Sublethal dosesof ß-lap cause consistently elevated OCRs over time. Both responses areNQO1-mediated. B, addition of MeOX enhances OCR rates in combinationwith sublethal doses of ß-lap, similar to a lethal dose of ß-lapachone(4 μM), presumably because of the loss of NAD+ and ATP derived fromPARP1 hyperactivation.

FIGS. 28A-B. NQO1 bioactivatable drug treatment suppresses glycolysis inMia PaCa2 cells. Both glucose utilization (A) and lactate production (B)are suppressed.

FIG. 29. A549 NSCLC cells were co-treated with 12 mM Methoxyamine (MeOX)and DNQ 87 for 2 h and relative survival was measured (as in Huang etal., Cancer Res., 2012).

FIGS. 30A-D. PARP1 inhibition enhances lethality of NQO1 bioactivatabledrugs; (A) AZD2281; (B) AG14361; (C) AG-014699; (D) BSI-201.

FIGS. 31A-F. Determination of nonlethal doses of PARP1 inhibitors alonein A549 NSCLC cells; (A) AZD-2281; (B) AG14361; (C) AG014699; (D)BSI-201; (E) ABT-888; (F) INO-1001.

FIGS. 32A-D. Data for the PARP1 inhibitor, AG014699 (rucaparib); (A)AG014699; (B) β-lap and AG014699; (C) AG014699; (D)β-lap and AG014699.

FIGS. 33A-D. AG014699 enhances the NQO1-dependent lethality ofß-lapachone in A549 NSCLC cells. A, control to demonstrate syntheticlethality of drug alone against a BRCA1−/−CAPAN-1 cells, whereas otherBRAC1 wild-type cancer cells (A549 and Mia PaCa-2 cells are completelyresistant to the PARP1 inhibitor. B, Demonstration that AG014699inhibits PARP1 hyperactivation in response to ß-lap. C, Synergisticlethality of AG014699 in combination with ß-lap, which is prevented withthe NQO1 inhibitor, dicoumarol. D, Dose-response of AG014699 incombination with ß-lap, and reversal by dicoumarol addition.

FIGS. 34A-D. PARP1 knockdown enhances lethality of ß-lapachone in triplenegative MDA-MB-231 (231) breast cancer cells; (A) 231 NQO1+/−; (B)ns-shRNA and PARP1-shRNA; (C) surviving fraction of MD-MBA-231; (D)surviving fraction of MD-MBA-231 NQO1+.

FIGS. 35A-B. PARP1 knockdown in MCF-7 breast cancer cells also enhancesß-lap lethality; (A) ß-lap at 5 μM; (B) relative survival of MCF-7cells.

FIG. 36. NQO1+H596 NSCLC cells treated with nontoxic doses ofß-lap+nontoxic doses of AG014699 show evidence of apoptosis in the formof cleaved caspase-3.

FIGS. 37A-B. PARP1 shRNA stable knockdown enhances lethality (A) butsuppresses ATP loss (B) due to the suppression (loss) of PARP1hyperactivation.

FIGS. 38A-B. ß-Lap-induced ATP loss is prevented with PARP1 shRNA stableknockdown or addition of AG014699; (A) MCF-7; (B) MCF-7-PARP1-shRNA.

FIGS. 39A-D show studies in Mia Paca-2 and DNQ combined PARP1 inhibitordata (A-D).

FIGS. 40-42 show studies in Mia Paca-2 and some DNQ combined PARP1inhibitor data.

FIGS. 43A-D. Pharmacokinetic analysis and target valuation of dC₃micelles in vivo; (A) dC3 and β-lap; (B) dC3 and β-lap; (C) comparisonof blank micelles vs. dC3; (D) relative ATP levels. dC3 is shown in thestructure below:

FIG. 44. DNQ Derivative PAR-PARP1 Formation in vivo.

FIG. 45. A549 treatment with DNQ-87 and a PARP inhibitor (AG014699).

FIGS. 46A-B. DNA damage via combinatorial treatment. (A) A549 NSCLCcells were pre-treated with or without Rucaparib (AG014699) and thenexposed to a nontoxic dose of ß-lapachone (3 μM) at the concentrationsindicated with or without the same PARP1 inhibitor. Cells were alsoexposed to a lethal dose of ß-lapachone (8 μM). Cells were then analyzedby neutral comet assays and DNA damage graphed in (B). (B) Data showthat DNA damage is dramatically increased in cells exposed to sublethaldoses of s-lap in the presence of PARP1 inhibitors, equivalent to DNAlesions created by a lethal dose of ß-lapachone.

FIGS. 47A-G. NQO1/Catalase ratios are elevated in breast cancer tumors,but low in associated normal tissue; (A-F) patient sample counts andexpression data; (G) NQO1 expression.

FIGS. 48A-E. NQO1 levels are also elevated in non-small cell lungcancers (NSCLCs) versus associated normal tissue (n=105) (A), whereasCatalase is elevated in Normal tissue and lower levels are found inNSCLC tumors (B). (C)-(E) expression and sample counts.

FIG. 49. NQO1/Catalase ratios in tumor versus normal tissue in humanNSCLC patient samples.

DETAILED DESCRIPTION

Tumor-selectivity remains a challenge for efficacious chemotherapeuticstrategies against cancer. Although the recent development ofβ-lapachone to specifically exploit elevated levels of NAD(P)H:quinoneoxidoreductase 1 (NQO1) in most solid tumors represents a novelchemotherapeutic approach, additional therapies that kill by variousmechanisms such as programmed necrosis at increased potency are needed.Deoxynyboquinone (DNQ) kills a wide spectrum of cancer cell types (i.e.,breast, non-small-cell lung, prostate, pancreatic) in an NQO1-dependentmanner with greatly improved (20- to 100-fold) potency compared toβ-lapachone. DNQ lethality relies on NQO1-dependent futile redoxcycling, using oxygen and generating extensive reactive oxygen species(ROS), particularly superoxide and hydrogen peroxide. Elevated ROSlevels cause extensive DNA lesions and PARP-1 hyperactivation that, inturn, results in severe NAD⁺/ATP depletion that stimulatescalcium-dependent programmed necrotic cell death responses unique tothis class of NQO1 ‘bioactivated’ drugs (e.g., β-lapachone and DNQ). Itwas surprisingly discovered that the combination of NQO1 bioactivatabledrugs and DNA repair inhibitors provide a synergistic andtumor-selective therapy.

Pancreatic cancer will be the second leading cause of cancer-relateddeaths in the U.S. by 2020, where 5-year survival is <6%. Currentstandard of care therapies offer little selectivity and high toxicity.Novel, tumor-selective approaches are therefore desperately needed.Nearly 90% of pancreatic cancers have elevated levels (10- to 40-fold)NQO1 and we recently showed that beta-lapachone (beta-lap) wasefficacious against pancreatic cancers in an NQO1-dependent manner (Liet al., Clin. Cancer Res., 2011). Beta-Lap is reduced by NQO1 like mostquinones, but unlike most, its hydroquinone form is unstable andspontaneously redox cycles in a futile manner where one mole of beta-lapgenerates ^(˜)120 moles of superoxide in two minutes, inducingpredominately DNA base and single strand break (SSB) damage. Thisresults in PARP1 hyperactivation and programmed necrosis, killing NQO1+cancer cells independent of: i, p53; ii, cell cycle; iii, all knownoncogenic drivers; and iv, apoptotic/antiapoptotic gene expression(e.g., Bax, Bak, Bcl2). Thus, ‘NQO1 bioactivatable drugs’ aretumor-selective and excellent candidates for improving efficacy ofcancer therapies including therapy for pancreatic cancer and other solidtumor cancers.

To improve its efficacy, the synergistic effects of adding the APsite-modifying drug and base excision repair (BER) inhibitor,methoxyamine (MeOX), with beta-lap against NQO1 over-expressingpancreatic cancer cells were examined. MeOX+beta-lap synergy resulted inenhanced lethality of sub-lethal doses of beta-lap; increased DNA lesionformation only in tumor cells; dramatic losses in ATP levels; anddramatic suppression of glycolysis. MeOX therefore enhances PARP1hyperactivation and synergistic cell killing by beta-lap.Mechanistically, the data indicate that PARP1 detects MeOX-AP modifiedsites or SSBs, allowing PARP1 hyperactivation and synergistic celldeath. Because MeOX is a nontoxic agent, the combination of MeOX and asecond agent can provide therapies for the treatment of cancers,particularly pancreatic cancer, as well as other NQO1 overexpressingsolid cancers.

Therapeutic Quinones

DNQ is a potent chemotherapeutic agent exhibiting a wide therapeuticwindow that holds great promise for targeted therapy against a widespectrum of difficult to treat cancers, including pancreatic andnon-small cell lung cancer. Despite considerable advances in cancerchemotherapy, the lack of selectivity of most cancer chemotherapeuticsremains a major limiting factor. Elevated NAD(P)H:quinoneoxidoreductase-1 (NQO1, DT-diaphorase, EC 1.6.99.2) levels found in mostsolid tumors, particularly in non-small-cell lung cancer cells (NSCLC),prostate, pancreatic and breast, provide a target for therapeutictreatments described herein. NQO1 is an inducible Phase II detoxifyingtwo-electron oxidoreductase capable of reducing most quinones, formingstable hydroquinones. In most cases, glutathione transferase thendetoxifies hydroquinones, conjugating them with glutathione forsecretion, and effectively avoiding more toxic semiquinones.

For some rare compounds, however, NQO1-mediated bioreduction can beexploited for antitumor activity. Rather than promoting detoxification,NQO1 activity can convert specific quinones into highly cytotoxicspecies. Most antitumor quinones dependent on NQO1 are DNA alkylators:(a) mitomycin C (MMC); (b) RH1; (c) E09; and (d) AZQ. However, these DNAalkylators are not only subject to detoxification pathways, butresistance from elevated or inducible DNA repair pathways limit theirusefulness. Furthermore, many of these drugs are efficient substratesfor one-electron oxidoreductases ubiquitously expressed in normaltissues.

The ortho-naphthoquinone, β-lapachone (β-lap, Scheme 1), kills culturedcancer cells and murine xenograft and orthotopic human or mouse tumormodels in vivo in an NQO1-dependent manner. In contrast to alkylatingquinones, β-lap induces cell death by NQO1-dependent reactive oxygenspecies (ROS) formation and oxidative stress. NQO1 metabolism of β-lapresults into an unstable hydroquinone that is spontaneously oxidized bytwo equivalents of dioxygen, generating superoxide.

A futile cycle of oxidoreduction is thus established, and elevatedsuperoxide levels, in turn cause massive DNA base and single strandbreak (SSBs) lesions that normally are easily and rapidly repaired.However, extensive DNA lesions created in β-lap-treated NQO1over-expressing cancer cells results in hyperactivation ofpoly(ADP-ribose)polymerase-1 (PARP1), an otherwise essential base andSSB repair enzyme. In turn, PARP1 hyperactivation results in dramaticreduction of the NAD⁺/ATP pool due to ADP-ribosylation, causingtremendous energy depletion and cell death. As a result, β-lap killsNQO1+ cancer cells by a unique programmed necrosis mechanism that is:(a) independent of caspase activation or p53 status; (b) independent ofbcl-2 levels; (c) not affected by BAX/BAK deficiencies; (d) independentof EGFR, Ras or other constitutive signal transduction activation;and/or (e) not dependent on proliferation, since NQO1 is expressed inall cell cycle phases. Thus, β-lap is an attractive experimentalchemotherapeutic, and various β-lap formulations have been, or are in,phase I/II clinical trials.

Deoxynyboquinone (DNQ, Scheme 1) is a promising anti-neoplastic agent.Prior data indicated that DNQ kills cancer cells through oxidativestress and ROS formation. The cytotoxicity of DNQ was partiallyprevented by N-acetylcysteine, a global free radical scavenger andprecursor to glutathione. It has now been show that DNQ undergoes anNQO1-dependent futile cycle similar to β-lap, where oxygen is consumed,and ROS is formed and extensive DNA damage triggers PARP1hyperactivation, with dramatic decreases in essential NAD⁺/ATPnucleotide pools, indicative of programmed necrosis. Importantly, DNQ is20- to 100-fold more potent than β-lap, with a significantly enhancedtherapeutic window in NQO1+ versus NQO1-NSCLC cells. EfficaciousNQO1-dependent killing by DNQ is also shown in breast, prostate, andpancreatic cancer models in vitro. Furthermore, in vitro NQO1 processesDNQ much more efficiently than β-lap, indicating that increasedutilization accounts for its increased potency. Thus, DNQ offerssignificant promise as a selective chemotherapeutic agent for thetreatment of solid tumors with elevated NQO1 levels, however, thecombination therapy described herein can provide efficacious therapieswith a variety of quinone compounds due to the synergy of thecombination.

Because NQO1 is overexpressed in the majority of solid tumors, and thecytotoxicity of the various quinone compounds depends predominately onthe elevated expression of the enzyme NQO1, thus the quinine compoundsand their derivatives can be excellent means to approach targeting solidtumors. The invention provides numerous new cytotoxic compounds that canbe used as new cancer therapeutics, as described herein.

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures. Furtherembodiments, forms, features, aspects, benefits, objects, and advantagesof the present application shall become apparent from the detaileddescription and figures provided herewith.

Use of NQO 1 Bioactivatable Drugs for Tumor-Specific use with DNA RepairInhibitors

NQO1 bioactivatable drugs (all β-lapachone and DNQ derivatives that aresubstrates for NQO1) generate tremendous levels of reactive oxygenspecies in an NQO1-dependent, tumor-selective manner, allowing the useof DNA repair inhibitors, including all PARP1 inhibitors, DNA doublestrand break repair inhibitors, as well as base excision repairinhibitors, to be used in a tumor-specific manner, effecting atumor-selective efficacy of both agents. DNA repair inhibitors, ingeneral, have failed because of the lack of tumor selectivity. Becausethese NQO1 bioactivatable drugs cause tumor selective production of DNAlesions, including DNA base damage, single strand breaks and doublestrand breaks, DNA repair inhibitors can be used to provide tumorselective antitumor activity. Tumor-selective activity and responsesinclude dramatic inhibition of glycolysis as well as othertumor-selective metabolism inhibition.

NQO1 bioactivatable drugs can be used to make DNA repair inhibitorstumor-selective in a manner that is not obvious unless one knows the DNAlesions generated, and in a manner that causes metabolic changes andcell death responses that are not obvious and are altered depending onthe DNA repair inhibitors used. For example, PARP1 inhibitorsadministered with DNQ bioactivatable drugs cause standard apoptoticresponses without energy losses. In contrast, DNA double strand breakrepair, single strand break repair, and base excision repair inhibitorsenhance PARP1 hyperactivation, with subsequent losses in energymetabolism and programmed necrosis.

The only current use of DNA repair inhibitors, such as PARP-1inhibitors, is through the unique exploitation of tumor-specificsynthetic lethality responses (e.g., use of P ARP1 inhibitors inBRACA1/2 mutant tumors). This, however, is a very limited use of DNArepair inhibitors—approximately only 5% of breast cancers only. Incontrast, the approach described herein can treat all cancers havingelevated NQO1 and lowered Catalase levels, while normal tissue haveelevated Catalase and low levels of NQO1. The methods described hereinprovide a new use of DNA repair inhibitors, allowing for their use in atumor-selective manner, while also potentiating NQO1 bioactivatabledrugs. Both agents can be used at nontoxic doses to render synergistic,tumor-selective efficacy responses.

To date, DNA repair inhibitors fail in the lack of tumor-selectiveresponses and efficacy. The methods described herein resolve theselimitations, while greatly potentiating NQO1 bioactivatable drugs. Themethods also allow the use of 2- to >4-fold lower doses of NQO1bioactivatable drugs, resolving toxic effects of these NQO1bioactivatable drugs (e.g., methemaglobinemia). In the therapeuticmethods, the inhibitors can be added before and after NQO1bioactivatable drugs, at a minimum before, and optionally before,simultaneously, after, or a combination thereof. Cell death responsesdepend on inhibitor used. The responses are not obvious and would entailspecific biomarkers to follow in vivo.

Definitions

In general, the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. Suchart-recognized meanings may be obtained by reference to technicaldictionaries, such as Hawley's Condensed Chemical Dictionary 14^(th)Edition, by R. J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

BER, base excision repair; SSBR, single strand break repair; DSBR,double strand break repair.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds, so that acompound X includes a plurality of compounds X. It is further noted thatthe claims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely,” “only,” and the like, inconnection with the recitation of claim elements or use of a “negative”limitation.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrase “one or more” is readily understood by one of skill in the art,particularly when read in context of its usage. For example, one or moresubstituents on a phenyl ring refers to one to five, or one to four, forexample if the phenyl ring is disubstituted.

The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer at each end of the range. Unless indicatedotherwise herein, the term “about” is intended to include values, e.g.,weight percentages, proximate to the recited range that are equivalentin terms of the functionality of the individual ingredient, thecomposition, or the embodiment.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements.

While the present invention can take many different forms, for thepurpose of promoting an understanding of the principles of theinvention, reference will now be made to the embodiments illustrated inthe drawings and specific language will be used to describe the same. Itwill nevertheless be understood that no limitation of the scope of theinvention is thereby intended. Any alterations and further modificationsof the described embodiments and any further applications of theprinciples of the invention as described herein are contemplated aswould normally occur to one skilled in the art to which the inventionrelates.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, including anyisomers, enantiomers, and diastereomers of the group members, aredisclosed separately. When a Markush group or other grouping is usedherein, all individual members of the group and all combinations andsub-combinations possible of the group are intended to be individuallyincluded in the disclosure. When a compound is described herein suchthat a particular isomer, enantiomer or diastereomer of the compound isnot specified, for example, in a formula or in a chemical name, thatdescription is intended to include each isomers and enantiomer of thecompound described individual or in any combination. Additionally,unless otherwise specified, all isotopic variants of compounds disclosedherein are intended to be encompassed by the disclosure. For example, itwill be understood that any one or more hydrogens in a moleculedisclosed can be replaced with deuterium or tritium. Isotopic variantsof a molecule are generally useful as standards in assays for themolecule and in chemical and biological research related to the moleculeor its use. Methods for making such isotopic variants are known in theart. Specific names of compounds are intended to be exemplary, as it isknown that one of ordinary skill in the art can name the same compoundsdifferently.

Whenever a range is given in the specification, for example, atemperature range, a time range, a carbon chain range, or a compositionor concentration range, all intermediate ranges and subranges, as wellas all individual values included in the ranges given are intended to beindividually included in the disclosure. It will be understood that anysubranges or individual values in a range or subrange that are includedin the description can be optionally excluded from embodiments of theinvention.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be replaced with either of the other two terms. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein.

A “chemotherapeutic agent” refers to any substance capable of reducingor preventing the growth, proliferation, or spread of a cancer cell, apopulation of cancer cells, tumor, or other malignant tissue. The termis intended also to encompass any antitumor or anticancer agent.

A “therapeutically effective amount” of a compound with respect to thesubject method of treatment refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a mammal, such as a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

The terms “treating”, “treat” and “treatment” include (i) preventing adisease, pathologic or medical condition from occurring (e.g.,prophylaxis); (ii) inhibiting the disease, pathologic or medicalcondition or arresting its development; (iii) relieving the disease,pathologic or medical condition; and/or (iv) diminishing symptomsassociated with the disease, pathologic or medical condition. Thus, theterms “treat”, “treatment”, and “treating” can extend to prophylaxis andcan include prevent, prevention, preventing, lowering, stopping orreversing the progression or severity of the condition or symptoms beingtreated. As such, the term “treatment” can include medical, therapeutic,and/or prophylactic administration, as appropriate. The term “treating”or “treatment” can include reversing, reducing, or arresting thesymptoms, clinical signs, and underlying pathology of a condition inmanner to improve or stabilize a subject's condition.

The terms “inhibit”, “inhibiting”, and “inhibition” refer to theslowing, halting, or reversing the growth or progression of a disease,infection, condition, or group of cells. The inhibition can be greaterthan about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, comparedto the growth or progression that occurs in the absence of the treatmentor contacting.

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity, including at the cellularor molecular level, for example, to bring about a physiologicalreaction, a chemical reaction, or a physical change, e.g., in asolution, in a reaction mixture, in vitro, or in vivo.

The term “exposing” is intended to encompass definitions as broadlyunderstood in the art. In an embodiment, the term means to subject orallow to be subjected to an action, influence, or condition. For exampleand by way of example only, a cell can be subjected to the action,influence, or condition of a therapeutically effective amount of apharmaceutically acceptable form of a chemotherapeutic agent.

The term “cancer cell” is intended to encompass definitions as broadlyunderstood in the art. In an embodiment, the term refers to anabnormally regulated cell that can contribute to a clinical condition ofcancer in a human or animal. In an embodiment, the term can refer to acultured cell line or a cell within or derived from a human or animalbody. A cancer cell can be of a wide variety of differentiated cell,tissue, or organ types as is understood in the art.

The term “tumor” refers to a neoplasm, typically a mass that includes aplurality of aggregated malignant cells.

The following groups can be R groups or bridging groups, as appropriate,in the formulas described herein.

The term “alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain preferably having from 1 to 30 carbon atoms.Short alkyl groups are those having 1 to 12 carbon atoms includingmethyl, ethyl, propyl, butyl, pentyl, and hexyl groups, including allisomers thereof. Long alkyl groups are those having 12-30 carbon atoms.The group may be a terminal group or a bridging group.

Alkyl, heteroalkyl, aryl, heteroaryl, and heterocycle groups, and cyclicand/or unsaturated versions thereof, can be R groups of Formula I, andeach group can be optionally substituted.

The term “substituted” indicates that one or more hydrogen atoms on thegroup indicated in the expression using “substituted” is replaced with a“substituent”. The number referred to by ‘one or more’ can be apparentfrom the moiety one which the substituents reside. For example, one ormore can refer to, e.g., 1, 2, 3, 4, 5, or 6; in some embodiments 1, 2,or 3; and in other embodiments 1 or 2. The substituent can be one of aselection of indicated groups, or it can be a suitable group known tothose of skill in the art, provided that the substituted atom's normalvalency is not exceeded, and that the substitution results in a stablecompound. Suitable substituent groups include, e.g., alkyl, alkenyl,alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, aroyl,(aryl)alkyl (e.g., benzyl or phenylethyl), heteroaryl, heterocycle,cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino,trifluoromethyl, trifluoromethoxy, trifluoromethylthio, difluoromethyl,acylamino, nitro, carboxy, carboxyalkyl, keto, thioxo, alkylthio,alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl,heteroarylsulfinyl, heteroarylsulfonyl, heterocyclesulfinyl,heterocyclesulfonyl, phosphate, sulfate, hydroxyl amine, hydroxyl(alkyl)amine, and cyano. Additionally, suitable substituent groups canbe, e.g., —X, —R, —O⁻, —OR, —SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN,—SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃, —NC(═O)R, —C(═O)R, —C(═O)NRR,—S(═O)₂O⁻, —S(═O)₂OH, —S(═O)₂R, —OS(═O)₂OR, —S(═O)₂NR, —S(═O)R,—OP(═O)O₂RR, —P(═O)O₂RR, —P(═O)(O⁻)₂, —P(═O)(OH)₂, —C(═O)R, —C(═O)X,—C(S)R, —C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR,or —C(NR)NRR, where each X is independently a halogen (“halo”): F, Cl,Br, or I; and each R is independently H, alkyl, aryl, (aryl)alkyl (e.g.,benzyl), heteroaryl, (heteroaryl)alkyl, heterocycle, heterocycle(alkyl),or a protecting group. As would be readily understood by one skilled inthe art, when a substituent is keto (═O) or thioxo (═S), or the like,then two hydrogen atoms on the substituted atom are replaced. In someembodiments, one or more of the substituents above can be excluded fromthe group of potential values for substituents on a substituted group.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, often having from 2to 14 carbons, or 2 to 10 carbons in the chain, including at least onecarbon atom and at least one heteroatom selected from the groupconsisting of O, N, P, Si and S, and wherein the nitrogen and sulfuratoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N, P and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. The heteroalkyl group can have, for example, one to about 20carbon atoms in a chain. Examples include, but are not limited to,—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂—CH₃, and —CN.Additional examples of heteroalkyl groups include alkyl ethers,secondary and tertiary alkyl amines, amides, alkyl sulfides, and thelike. The group may be a terminal group or a bridging group. As usedherein, reference to a chain when used in the context of a bridginggroup refers to the direct chain of atoms linking the two terminalpositions of the bridging group.

The term “alcohol” as used herein may be defined as an alcohol thatcomprises a C₁₋₁₂ alkyl moiety substituted at a hydrogen atom with onehydroxyl group. Alcohols include ethanol, n-propanol, i-propanol,n-butanol, i-butanol, s-butanol, t-butanol, n-pentanol, i-pentanol,n-hexanol, cyclohexanol, n-heptanol, n-octanol, n-nonanol, n-decanol,and the like. The carbon atoms in alcohols can be straight, branched orcyclic.

“Acyl” may be defined as an alkyl-CO— group in which the alkyl group isas described herein. Examples of acyl include acetyl and benzoyl. Thealkyl group can be a C₁-C₆ alkyl group. The group may be a terminalgroup or a bridging (i.e., divalent) group.

“Alkoxy” refers to an —O-alkyl group in which alkyl is defined herein.Preferably the alkoxy is a C₁-C₆alkoxy. Examples include, but are notlimited to, methoxy and ethoxy. The group may be a terminal group or abridging group.

“Alkenyl” as a group or part of a group denotes an aliphatic hydrocarbongroup containing at least one carbon-carbon double bond and which may bestraight or branched preferably having 2-14 carbon atoms, morepreferably 2-12 carbon atoms, most preferably 2-6 carbon atoms, in thenormal chain. The group may contain a plurality of double bonds in thenormal chain and the orientation about each is independently E or Z.Exemplary alkenyl groups include, but are not limited to, ethenyl,propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl. Thegroup may be a terminal group or a bridging group.

“Alkynyl” as a group or part of a group may be defined as an aliphatichydrocarbon group containing a carbon-carbon triple bond, the chain ofwhich may be straight or branched preferably having from 2-14 carbonatoms, more preferably 2-12 carbon atoms, more preferably 2-6 carbonatoms in the normal chain. Exemplary structures include, but are notlimited to, ethynyl and propynyl. The group may be a terminal group or abridging group.

“Alkenyloxy” refers to an —O— alkenyl group in which alkenyl is asdefined herein. Preferred alkenyloxy groups are C₁-C₆ alkenyloxy groups.The group may be a terminal group or a bridging group.

“Alkynyloxy” refers to an —O-alkynyl group in which alkynyl is asdefined herein. Preferred alkynyloxy groups are C₁-C₆ alkynyloxy groups.The group may be a terminal group or a bridging group.

“Alkoxycarbonyl” refers to an —C(O)—O-alkyl group in which alkyl is asdefined herein. The alkyl group is preferably a C₁-C₆ alkyl group.Examples include, but not limited to, methoxycarbonyl andethoxycarbonyl. The group may be a terminal group or a bridging group.

“Alkylsulfinyl” may be defined as a —S(O)-alkyl group in which alkyl isas defined above. The alkyl group is preferably a C₁-C₆ alkyl group.Exemplary alkylsulfinyl groups include, but not limited to,methylsulfinyl and ethylsulfinyl. The group may be a terminal group or abridging group.

“Alkylsulfonyl” refers to a —S(O)₂-alkyl group in which alkyl is asdefined above. The alkyl group is preferably a C₁-C₆ alkyl group.Examples include, but not limited to methylsulfonyl and ethylsulfonyl.The group may be a terminal group or a bridging group.

“Amino” refers to —NH₂, and “alkylamino” refers to —NR₂, wherein atleast one R is alkyl and the second R is alkyl or hydrogen. The term“acylamino” refers to RC(═O)NH—, wherein R is alkyl or aryl. The alkylgroup can be, for example, a C₁-C₆ alkyl group. Examples include, butare not limited to methylamino and ethylamino. The group may be aterminal group or a bridging group.

“Alkylaminocarbonyl” refers to an alkylamino-carbonyl group in whichalkylamino is as defined above. The group may be a terminal group or abridging group.

“Cycloalkyl” refers to a saturated or partially saturated, monocyclic orfused or spiro polycyclic, carbocycle of 3 to about 30 carbon atoms,often containing 3 to about 9 carbons per ring, such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and the like. Itincludes monocyclic systems such as cyclopropyl and cyclohexyl, bicyclicsystems such as decalin, and polycyclic systems such as adamantane. Thegroup may be a terminal group or a bridging group.

“Cycloalkenyl” may be defined as a non-aromatic monocyclic ormulticyclic ring system containing at least one carbon-carbon doublebond and preferably having from 5-10 carbon atoms per ring. Exemplarymonocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl orcycloheptenyl. The cycloalkenyl group may be substituted by one or moresubstituent groups. The group may be a terminal group or a bridginggroup.

Alkyl and cycloalkyl groups can be substituents on the alkyl portions ofother groups, such as without limitation, alkoxy, alkyl amines, alkylketones, arylalkyl, heteroarylalkyl, alkylsulfonyl and alkyl estersubstituents and the like. The group may be a terminal group or abridging group.

“Cycloalkylalkyl” may be defined as a cycloalkyl-alkyl-group in whichthe cycloalkyl and alkyl moieties are as previously described. Exemplarymonocycloalkylalkyl groups include cyclopropylmethyl, cyclopentylmethyl,cyclohexylmethyl and cycloheptylmethyl. The group may be a terminalgroup or a bridging group.

“Heterocycloalkyl” refers to a saturated or partially saturatedmonocyclic, bicyclic, or polycyclic ring containing at least oneheteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to3 heteroatoms in at least one ring. Each ring is preferably from 3 to 10membered, more preferably 4 to 7 membered. Examples of suitableheterocycloalkyl substituents include pyrrolidyl, tetrahydrofuryl,tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl,morpholino, 1,3-diazapane, 1,4-diazapane, 1,4-oxazepane, and1,4-oxathiapane. The group may be a terminal group or a bridging group.

“Heterocycloalkenyl” refers to a heterocycloalkyl as described above butcontaining at least one double bond. The group may be a terminal groupor a bridging group.

“Heterocycloalkylalkyl” refers to a heterocycloalkyl-alkyl group inwhich the heterocycloalkyl and alkyl moieties are as previouslydescribed. Exemplary heterocycloalkylalkyl groups include(2-tetrahydrofuryl)methyl, and (2-tetrahydrothiofuranyl)methyl. Thegroup may be a terminal group or a bridging group.

“Halo” refers to a halogen substituent such as fluoro, chloro, bromo, oriodo.

The term “aryl” refers to an aromatic hydrocarbon group derived from theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. The radical can be at a saturated or unsaturatedcarbon atom of the parent ring system. The aryl group can have from 6 to18 carbon atoms. The aryl group can have a single ring (e.g., phenyl) ormultiple condensed (fused) rings, wherein at least one ring is aromatic(e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Typicalaryl groups include, but are not limited to, radicals derived frombenzene, naphthalene, anthracene, biphenyl, and the like. The aryl canbe unsubstituted or optionally substituted, as described above for alkylgroups.

The term “heteroaryl” is defined herein as a monocyclic, bicyclic, ortricyclic ring system containing one, two, or three aromatic rings andcontaining at least one nitrogen, oxygen, or sulfur atom in an aromaticring, and which can be unsubstituted or substituted, for example, withone or more, and in particular one to three, substituents, as describedabove in the definition of “substituted”. Examples of heteroaryl groupsinclude, but are not limited to, 2H-pyrrolyl, 3H-indolyl,4H-quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl,β-carbolinyl, carbazolyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl,furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolisinyl,indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl,isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl,phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl,phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl,pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl,pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, tetrazolyl,and xanthenyl. In one embodiment the term “heteroaryl” denotes amonocyclic aromatic ring containing five or six ring atoms containingcarbon and 1, 2, 3, or 4 heteroatoms independently selected fromnon-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O,alkyl, aryl, or (C₁-C₆)alkylaryl. In another embodiment heteroaryldenotes an ortho-fused bicyclic heterocycle of about eight to ten ringatoms derived therefrom, particularly a benz-derivative or one derivedby fusing a propylene, trimethylene, or tetramethylene diradicalthereto.

The term “heterocycle” refers to a saturated or partially unsaturatedring system, containing at least one heteroatom selected from the groupoxygen, nitrogen, and sulfur, and optionally substituted with one ormore groups as defined herein under the term “substituted”. Aheterocycle can be a monocyclic, bicyclic, or tricyclic group containingone or more heteroatoms. A heterocycle group also can contain an oxogroup (═O) attached to the ring. Non-limiting examples of heterocyclegroups include 1,3-dihydrobenzofuran, 1,3-dioxolane, 1,4-dioxane,1,4-dithiane, 2H-pyran, 2-pyrazoline, 4H-pyran, chromanyl,imidazolidinyl, imidazolinyl, indolinyl, isochromanyl, isoindolinyl,morpholine, piperazinyl, piperidine, piperidyl, pyrazolidine,pyrazolidinyl, pyrazolinyl, pyrrolidine, pyrroline, quinuclidine, andthiomorpholine.

The abbreviation “DNQ_(d)” as used herein refers to an analog orderivative of DNQ.

Additional groups that can be bridging groups or terminal groups of R₁,R₂, R₃, and R₄ are described below.

The term “carbonate ester” may be defined as a functional group having ageneral structure R′OC(═O)OR, where R′ can be the tricyclic core ofFormula I and R can be as defined in the definitions of the variables ofFormula I.

The term “ester” may be defined as a functional group having a generalstructure RC(═O)OR′, where R′ can be the tricyclic core of Formula I andR can be as defined in the definitions of the variables of Formula I, orvice versa.

A “pyridyl” group can be a 2-pyridyl, 3-pyridyl, or 4-pyridyl group.

The term “sulfhydryl” may be defined as a functional group having ageneral structure —S—H.

The term “sulfinyl” may be defined as a functional group having ageneral structure R—S(═O)—R′, where R′ can be the tricyclic core ofFormula I and R can be as defined in the definitions of the variables ofFormula I, or vice versa.

The term “sulfonyl” may be defined as a functional group having ageneral structure R—S(═O)₂—R′, where R′ can be the tricyclic core ofFormula I and R can be as defined in the definitions of the variables ofFormula I, or vice versa.

The term “hexose” may be defined as a monosaccharide having six carbonatoms having the general chemical formula C₆H₁₂O₆ and can includealdohexoses which have an aldehyde functional group at position 1 orketohexoses which have a ketone functional group at position 2. Examplealdohexoses include, allose, altrose, glucose, mannose, gulose, idose,galactose, and talose, in either D or L form.

PARP inhibitors are a group of pharmacological inhibitors of the enzymepoly ADP ribose polymerase (PARP). They are developed for multipleindications including the treatment of cancer. Several forms of cancerare more dependent on PARP than regular cells, making PARP an attractivetarget for cancer therapy. PARP-1 inhibitors are particularly useful inthe combination therapies described herein. PARP-1 inhibitors can bepurchased from commercial vendors such as Selleck Chemicals. Examples ofPARP-1 inhibitors include the inhibitors listed in Table 1 below.

TABLE 1 PARP-1 Inhibitors. AG-014699 ABT-888 *BSI-201 AZD2281 PARP1Inhibitor: (Rucaparib) AG14361 (Veliparib) (Iniparib) (Olaparib)INO-1001 Formulation: Normal 1% DMSO 0.9% NaCl HPBCD HPBCD water salineadj. to pH 4.0 **Sensitivity Enhancement (change in survival)β-Lapachone 10 5 10 3 10 7 Deoxynyboquinone 100 10 15 4 20 10 (DNQ)*Although BSI-201 was a reported PARP1 inhibitor and did synergize withβ-lap or DNQ, using PARP1 knockdown cells we noted additional synergy,which was not observed with the other PARP1 inhibitors. Furthermore, itsaddition did not prevent PARP1 PAR formation as did all for the otherinhibitors. We conclude, therefore that BSI-201 is not a PARP1inhibitor, but rather a DNA damaging agent and this explains the synergynoted. **Varying doses (μM) of each of the PARP1 inhibitors were addedwith varying doses of β-lapachone or DNQ. Reported sensitivity valuesrepresent changes in survival of A549 NSCLC cells or MiaPaCa-2pancreatic cancer cells at nontoxic doses of either β-lapachone (3 μMfor A549, 2 μM Mia Paca2 cells) or DNQ (0.02 μM for A549 cells, 0.025 μMfor MiaPaca-2 cells) in combination with an optimal dose (i.e., 15 μM)of each PARP1 inhibitor. A value of 10 equals one log kill, and a valueof 100 equals two logs kill and so on. PARP1 inhibitors were nonlethalto as high as 100 μM.Compounds and Methods of the Invention

The invention provides DNQ compounds, beta-lapachone and derivativesthereof, and the use of NQO1 bioactivatable drugs in combination therapyfor the treatment of cancer. Examples of DNQ compounds include compoundsof Formula (I):

wherein

R₁, R₂, R₃, and R₄ are each independently —H or —X—R;

each X is independently a direct bond or a bridging group, wherein thebridging group is —O—, —S—, —NH—, —C(═O)—, —O—C(═O)—, —C(═O)—O—,—O—C(═O)—O—, or a linker of the formula —W-A-W—, wherein

each W is independently —N(R′)C(═O)—, —C(═O)N(R′)—, —OC(═O)—, —C(═O)O—,—O—, —S—, —S(O)—, —S(O)₂—, —N(R′)—, —C(═O)—, —(CH₂)_(n)— where n is1-10, or a direct bond, wherein each R′ is independently H,(C₁-C₆)alkyl, or a nitrogen protecting group; and

each A is independently (C₁-C₂₀)alkyl, (C₂-C₁₆)alkenyl, (C₂-C₁₆)alkynyl,(C₃-C₈)cycloalkyl, (C₆-C₁₀)aryl, —(OCH₂—CH₂)_(n)— where n is 1 to about20, —C(O)NH(CH₂)_(n)— wherein n is 1 to about 6, —OP(O)(OH)O—,—OP(O)(OH)O(CH₂)_(n)— wherein n is 1 to about 6, or (C₁-C₂₀)alkyl,(C₂-C₁₆)alkenyl, (C₂-C₁₆)alkynyl, or —(OCH₂—CH₂)_(n)— interruptedbetween two carbons, or between a carbon and an oxygen, with acycloalkyl, heterocycle, or aryl group;

each R is independently alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl,(cycloalkyl)alkyl, (heterocycloalkyl)alkyl, (cycloalkyl)heteroalkyl,(heterocycloalkyl)heteroalkyl, aryl, heteroaryl, (aryl)alkyl,(heteroaryl)alkyl, hydrogen, hydroxy, hydroxyalkyl, alkoxy,(alkoxy)alkyl, alkenyloxy, alkynyloxy, (cycloalkyl)alkoxy,heterocycloalkyloxy, amino, alkylamino, aminoalkyl, acylamino,arylamino, sulfonylamino, sulfinylamino, —COR^(x), —COOR^(x),—CONHR^(x), —NHCOR^(x), —NHCOOR^(x), —NHCONHR^(x), —N₃, —CN, —NC, —NCO,—NO₂, —SH, -halo, alkoxycarbonyl, alkylaminocarbonyl, sulfonate,sulfonic acid, alkylsulfonyl, alkylsulfinyl, arylsulfonyl, arylsulfinyl,aminosulfonyl, R^(x)S(O)R^(y)—, R^(x)S(O)₂R^(y)—,R^(x)C(O)N(R^(x))R^(y)—, R^(x)SO₂N(R^(x))R^(y)—,R^(x)N(R^(x))C(O)R^(y)—, R^(x)N(R^(x))SO₂R^(y)—,R^(x)N(R^(x))C(O)N(R^(x))R^(y)—, carboxaldehyde, acyl, acyloxy, —OPO₃Z₂,—OPO₃Z₂ where Z is an inorganic cation, or saccharide; where each R^(x)is independently H, OH, alkyl or aryl, and each R^(y) is independently agroup W;

wherein any alkyl or aryl can be optionally substituted with one or morehydroxy, amino, cyano, nitro, or halo groups;

or a salt or solvate thereof.

In some embodiments, when R₁, R₂, and R₃ are methyl, R₄ is not H ormethyl. In other embodiments, when R₁, R₃, and R₄ are methyl, the group—X—R of R₂ is not —CH₂—OAc. In certain embodiments, when R₁, R₃, and R₄are methyl, the R group of R₂ is not acyloxy. In various embodiments,R₁-R₄ are not each H. In certain embodiments, R₁-R₄ are not each alkyl,such as unsubstituted alkyl. In some embodiments, R₁-R₄ are not eachmethyl.

In one embodiment, R₁, R₂, R₃, and R₄ are each (C₁₋₂₀)alkyl groups. Insome embodiments, the (C₁₋₂₀)alkyl group is a (C₂₋₂₀)alkyl group, a(C₃₋₂₀)alkyl group, a (C₄₋₂₀)alkyl group, a (C₅₋₂₀)alkyl group, or a(C₁₀₋₂₀)alkyl group. The alkyl groups can be substituted, for example,with a hydroxyl or phosphate group. The phosphate group can be aphosphonic acid or a phosphonic acid salt, such as a lithium salt, asodium salt, a potassium salt, or other known salt of phosphonic acids.

A specific value for R₁ is H. A specific value for R₂ is H. A specificvalue for R₃ is H. A specific value for R₄ is H.

A specific value for R₁ is methyl. A specific value for R₂ is methyl. Aspecific value for R₃ is methyl. A specific value for R₄ is methyl. Themethyl can be substituted as described above for the term “substituted”.

In some embodiments of Formula (I):

-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is2-methyl-propane;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is butyl;-   R₁ and R₄ are methyl and R₃ is hydrogen; and R₂ is ethyl;-   R₁ and R₂ are methyl and R₃ is hydrogen; and R₄ is ethyl;-   R₁ is methyl; R₃ is hydrogen; R₂ is propyl; and R₄ is butyl;-   R₁ and R₄ are methyl; R₂ is propyl and R₃ is hydrogen;-   R₁ is propyl; R₂ and R₄ are methyl and R₃ is hydrogen;-   R₁ and R₂ are ethyl; R₃ is hydrogen; and R₂ is methyl;-   R₁ is propyl; R₂ is methyl; R₃ is hydrogen; and R₄ is butyl;-   R₁ and R₂ are propyl; R₃ is hydrogen; and R₄ is butyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is C₁₂alkyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is tert-butyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is hydroxypropyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is 3,3-dimethylbutyl    [—CH₂CH₂C(CH₃)₂CH₃];-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is 3-methybutyl    [—CH₂CH₂CH(CH₃)CH₃];-   R₂ and R₄ are methyl; R₃ is hydrogen; and R₁ is ethyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is propyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is n-pentyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is n-hexyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is isopropyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is cyclooctyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is cyclopropyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is methylcyclopropyl;-   R₁ and R₂ are methyl; R₃ is hydrogen; and R₄ is ethylcyclopropyl;-   R₁ is C₁₂alkyl; R₂ and R₄ are methyl; and R₃ is hydrogen;-   R₁ and R₄ are methyl; R₃ is hydrogen; and R₂ is C₁₂alkyl;-   R₁, R₂, and R₃ are methyl; and R₄ is —CH₂OPO₃Na₂;-   R₁ is —CH₂OPO₃Na₂; R₂ and R₃ are methyl; and R₄ is hydrogen;-   R₁ and R₃ are methyl; R₂ is —CH₂OPO₃Na₂; and R₄ is hydrogen;-   R₁ and R₂ are methyl; R₃ is —CH₂OPO₃Na₂; and R₄ is hydrogen;-   R₁ and R₂ are methyl; R₃ is —CH₂CH₂OPO₃Na₂; and R₄ is hydrogen;-   R₁, R₂, and R₃ are methyl; and R₄ is —CH₂OH;-   R₁ is —CH₂OH; R₂ and R₃ are methyl; and R₄ is hydrogen;-   R₁ and R₃ are methyl; R₂ is —CH₂OH; and R₄ is hydrogen;-   R₁ and R₂ are methyl; R₃ is —CH₂OH; and R₄ is hydrogen; or-   R₁ and R₂ are methyl; R₃ is —CH₂CH₂OH; and R₄ is hydrogen.    Additional specific compounds and formulas of the invention are    illustrated in FIGS. 11 and 12.

In certain embodiments of Formula I, R¹ is (C₁₋₄) alkyl group. Incertain instances, R¹ is (C₁₋₃) alkyl group. In certain instances, R¹ is(C₁₋₂) alkyl group.

In certain embodiments of Formula I, R² is (C₁₋₄) alkyl group. Incertain instances, R² is (C₁₋₃) alkyl group. In certain instances, R² is(C₁₋₂) alkyl group.

In certain embodiments of Formula I, R³ is hydrogen.

In certain embodiments of Formula I, R⁴ is an optionally substituted(C₁₋₁₀) alkyl group, where the alkyl group is substituted with hydroxyl,halogen, amino, or thiol. In certain instances, R⁴ is (C₁₋₁₀) alkylgroup, (C₁₋₈) alkyl group, (C₁₋₆) alkyl group, or (C₁₋₄) alkyl group. Incertain instances, R⁴ is (C₂₋₆) alkyl group. In certain instances, R⁴ isa substituted (C₁₋₁₀) alkyl group, substituted (C₁₋₈) alkyl group,substituted (C₁₋₆) alkyl group, or substituted (C₁₋₄) alkyl group, wherethe alkyl group is substituted with hydroxyl, halogen, amino, or thiol.In certain instances, R⁴ is an alkyl group is substituted with hydroxyl.In certain instances, R⁴ is an alkyl group is substituted with halogen.In certain instances, R⁴ is an alkyl group is substituted with amino. Incertain instances, R⁴ is an alkyl group is substituted with thiol.

In certain embodiments of Formula I, R¹ and R² are independently (C₁₋₄)alkyl groups; R³ is hydrogen; and R⁴ is an optionally substituted(C₁₋₁₀) alkyl group, where the alkyl group is substituted with hydroxyl,halogen, amino, and thiol.

In certain embodiments of Formula I, R¹ and R² are independently (C₁₋₂)alkyl groups; R³ is hydrogen; and R⁴ is an optionally substituted(C₁₋₁₀) alkyl group, where the alkyl group is substituted with hydroxyl,halogen, amino, and thiol.

In certain embodiments of Formula I, R¹ and R² are independently (C₁₋₂)alkyl groups; R³ is hydrogen; and R⁴ is (C₁₋₁₀) alkyl group. In certainembodiments of Formula I, R¹ and R² are independently (C₁₋₂) alkylgroups; R³ is hydrogen; and R⁴ is (C₁₋₈) alkyl group. In certainembodiments of Formula I, R¹ and R² are independently (C₁₋₂) alkylgroups; R³ is hydrogen; and R⁴ is (C₁₋₆) alkyl group. In certainembodiments of Formula I, R¹ and R² are independently (C₁₋₂) alkylgroups; R³ is hydrogen; and R⁴ is (C₁₋₄) alkyl group. In certainembodiments of Formula I, R¹ and R² are independently (C₁₋₂) alkylgroups; R³ is hydrogen; and R⁴ is (C₂₋₆) alkyl group. In certainembodiments of Formula I, R¹ and R² are independently (C₁₋₂) alkylgroups; R³ is hydrogen; and R⁴ is a substituted (C₁₋₆) alkyl group,where the alkyl group is substituted with hydroxyl, halogen, amino, andthiol. In certain embodiments of Formula I, R¹ and R² are independently(C₁₋₂) alkyl groups; R³ is hydrogen; and R⁴ is a substituted (C₁₋₄)alkyl group, where the alkyl group is substituted with hydroxyl,halogen, amino, and thiol.

In certain embodiments, a compound of Formula I is Compound 87 or a saltor solvate thereof:

In certain embodiments, a compound of Formula I is Compound 9-253 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 9-251 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 10-41 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 109 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 107 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 9-281 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 9-249 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 9-255 or asalt or solvate thereof:

In certain embodiments, a compound of Formula I is Compound 9-257 or asalt or solvate thereof:

The invention also provides a pharmaceutical composition comprising acompound of Formula (I) and a pharmaceutically acceptable diluent,excipient, or carrier. The carrier can be water, for example, in thepresence of hydroxypropyl-β-cyclodextrin (HPβCD). The solubility of thecompound can be increase by about 100 times, about 200 times, about 500times, about 1000 times, about 2000 times, or about 3000 times, comparedto the compounds solubility in water without HPβCD. Additional DNQcompounds and methods are described by International Application No.PCT/US12/59988 (Hergenrother et al.).

As to any of the above formulas or groups that contain one or moresubstituents, it is understood, of course, that such groups do notcontain any substitution or substitution patterns that are stericallyimpractical and/or synthetically non-feasible. In addition, thecompounds of this invention include all stereochemical isomers arisingfrom the substitution of these compounds.

Selected substituents of the compounds described herein may be presentto a recursive degree. In this context, “recursive substituent” meansthat a substituent may recite another instance of itself. Because of therecursive nature of such substituents, theoretically, a large number maybe present in any given claim. One of ordinary skill in the art ofmedicinal chemistry and organic chemistry understands that the totalnumber of such substituents is reasonably limited by the desiredproperties of the compound intended. Such properties include, by ofexample and not limitation, physical properties such as molecularweight, solubility or log P, application properties such as activityagainst the intended target, and practical properties such as ease ofsynthesis.

Recursive substituents are an intended aspect of the invention. One ofordinary skill in the art of medicinal and organic chemistry understandsthe versatility of such substituents. To the degree that recursivesubstituents are present in a claim of the invention, the total numberwill be determined as set forth above. In some embodiments, recursivesubstituents are present only to the extent that the molecular mass ofthe compound is about 400 to about 1600, about 450 to about 1200, about500 to about 100, about 600 to about 800. In other embodiments,recursive substituents are present only to the extent that the molecularmass of the compound is less than 2000, less than 1800, less than 1600,less than 1500, less than 1400, less than 1200, less than 1000, lessthan 900, less than 800, less than 750, less than 700, or less thanabout 600.

Patients with solid tumors having elevated NQO1 levels can be treatedthrough the administration of an effective amount of a pharmaceuticallyactive form of DNQ and/or DNQ_(d) (DNQ compounds). DNQ and DNQ_(d)compounds can be, for example, a compound defined by one of the formulasof FIG. 11, or a compound illustrated in FIG. 12. In FIG. 11 wheren=1-30, the value of n can be 1 or any integer from 1 up to about 30.Thus, the range 1-30 includes each individual integer from 1 to 30 andany ranges from any one to any second number from 1 to 30. In each rangedescribed herein, a portion of the range may also be excluded from theembodiment defined. For example, in various embodiments, a variable ncan be 6-24, and another n variable of the same formula can be 1-24.

In FIG. 11 for DNQ_(d)-20, R₁, R₂, and R₃ can be as defined for FormulaI above. In various embodiments, R₁, R₂, and R₃ can also eachindependently be C₁₋₂₀alkyl, or each of R₁, R₂ or R₃ can beindependently be linked to the anomeric position of a hexose, optionallythrough a linker, such as a linker of formula —W-A-W— or a(C₁-C₁₀)alkylene group.

In FIG. 11 for DNQ_(d)-27 and DNQ_(d)-28, X can be a linker of formula—W-A-W— or a divalent bridging group such as a divalent alkyl, alkenyl,alkynyl, heteroalkyl, acycloalkyl, cycloalkenyl, heterocycloalkyl,heterocycloalkenyl, cycloalkylalkyl, heterocycloalkylalkyl,cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, alkoxy, alkoxyalkyl,alkenyloxy, alkynyloxy, cycloalkylkoxy, heterocycloalkyloxy, amino,alkylamino, aminoalkyl, acylamino, arylamino, sulfonylamino,sulfinylamino, alkoxycarbonyl, alkylaminocarbonyl, sulfonyl,alkylsulfonyl, alkylsulfinyl, arylsulfonyl, arylsulfinyl, aminosulfonyl,or acyl, each of which may be optionally substituted.

In FIG. 11 for DNQ_(d)-29, each X can independently be a linker offormula —W-A-W— or a divalent bridging group as described above forDNQ_(d)-27 and DNQ_(d)-28; and each Y can independently be:

 (1) Hydroxyl,  (2) Aldehyde,  (3) Carboxyl,  (4) Haloformyl,  (5)Hydroperoxy,  (6) Phenyl,  (7) Benzyl,  (8) Alkyl,  (9) Alkenyl, (10)Alkynyl, (11) Acetate, (12) Amino, (13) Azide, (14) Azo, (15) Cyano,(16) Isocyanato, (17) Nitrate, (18) Isonitrile, (19) Nitrosooxy, (20)Nitro, (21) Nitroso, (22) Pyridyl, (23) Sulfhydryl, (24) Sulfonic acid,(25) Sulfonate, (26) Isothiocyanato, (27) Phosphine, (28) Phosphate,(29) Halo, or (30) Hexose.

The invention also provides methods of treating a patient that has tumorcells having elevated NQO1 levels. The methods can include administeringto a patient having tumor cells with elevated NQO1 levels atherapeutically effective amount of a compound of Formula (I), or acomposition described herein. The invention further provides methods oftreating a tumor cell having an elevated NQO1 level comprising exposingthe tumor cell to a therapeutically effective amount of a compound orcomposition described herein, wherein the tumor cell is treated, killed,or inhibited from growing. The tumor or tumor cells can be malignanttumor cells. In some embodiments, the tumor cells are cancer cells, suchas Non-Small-Cell Lung Carcinoma.

The methods of the invention may be thus used for the treatment orprevention of various neoplasia disorders including acral lentiginousmelanoma, actinic keratoses, adenocarcinoma, adenoid cycstic carcinoma,adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors,bartholin gland carcinoma, basal cell carcinoma, bronchial glandcarcinomas, capillary, carcinoids, carcinoma, carcinosarcoma, cavernous,cholangiocarcinoma, chondosarcoma, choriod plexus papilloma/carcinoma,clear cell carcinoma, cystadenoma, endodermal sinus tumor, endometrialhyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma,ependymal, epitheloid, Ewing's sarcoma, fibrolamellar, focal nodularhyperplasia, gastrinoma, germ cell tumors, glioblastoma, glucagonoma,hemangiblastomas, hemangioendothelioma, hemangiomas, hepatic adenoma,hepatic adenomatosis, hepatocellular carcinoma, insulinoma,intaepithelial neoplasia, interepithelial squamous cell neoplasia,invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma,lentigo maligna melanomas, malignant melanoma, malignant mesothelialtumors, medulloblastoma, medulloepithelioma, melanoma, meningeal,mesothelial, metastatic carcinoma, mucoepidermoid carcinoma,neuroblastoma, neuroepithelial adenocarcinoma nodular melanoma, oat cellcarcinoma, oligodendroglial, osteosarcoma, pancreatic polypeptide,papillary serous adenocarcinoma, pineal cell, pituitary tumors,plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma,retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cellcarcinoma, soft tissue carcinomas, somatostatin-secreting tumor,squamous carcinoma, squamous cell carcinoma, submesothelial, superficialspreading melanoma, undifferentiated carcinoma, uveal melanoma,verrucous carcinoma, vipoma, well differentiated carcinoma, and Wilm'stumor. Accordingly, the compositions and methods described herein can beused to treat bladder cancer, brain cancer (including intracranialneoplasms such as glioma, meninigioma, neurinoma, and adenoma), breastcancer, colon cancer, lung cancer (SCLC or NSCLC) ovarian cancer,pancreatic cancer, and prostate cancer.

Methods of Making the Compounds of the Invention

The invention also relates to methods of making the compounds andcompositions of the invention. The compounds and compositions can beprepared by any of the applicable techniques of organic synthesis. Manysuch techniques are well known in the art. However, many of the knowntechniques are elaborated in Compendium of Organic Synthetic Methods(John Wiley & Sons, New York), Vol. 1, Ian T. Harrison and ShuyenHarrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol.3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade, Jr.,1980; Vol. 5, Leroy G. Wade, Jr., 1984; and Vol. 6, Michael B. Smith; aswell as standard organic reference texts such as March's AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure, 5^(th) Ed. byM. B. Smith and J. March (John Wiley & Sons, New York, 2001),Comprehensive Organic Synthesis; Selectivity, Strategy & Efficiency inModern Organic Chemistry, in 9 Volumes, Barry M. Trost, Ed.-in-Chief(Pergamon Press, New York, 1993 printing)); Advanced Organic Chemistry,Part B: Reactions and Synthesis, Second Edition, Cary and Sundberg(1983); Protecting Groups in Organic Synthesis, Second Edition, Greene,T. W., and Wutz, P. G. M., John Wiley & Sons, New York; andComprehensive Organic Transformations, Larock, R. C., Second Edition,John Wiley & Sons, New York (1999).

A number of exemplary methods for the preparation of the compositions ofthe invention are provided below. These methods are intended toillustrate the nature of such preparations are not intended to limit thescope of applicable methods. Additional methods and useful techniquesare described in WO 2013/056073 (Hergenrother et al.).

Generally, the reaction conditions such as temperature, reaction time,solvents, work-up procedures, and the like, will be those common in theart for the particular reaction to be performed. The cited referencematerial, together with material cited therein, contains detaileddescriptions of such conditions. Typically the temperatures will be−100° C. to 200° C., solvents will be aprotic or protic depending on theconditions required, and reaction times will be 1 minute to 10 days.Work-up typically consists of quenching any unreacted reagents followedby partition between a water/organic layer system (extraction) andseparation of the layer containing the product. Oxidation and reductionreactions are typically carried out at temperatures near roomtemperature (about 20° C.), although for metal hydride reductionsfrequently the temperature is reduced to 0° C. to −100° C. Heating canalso be used when appropriate. Solvents are typically aprotic forreductions and may be either protic or aprotic for oxidations. Reactiontimes are adjusted to achieve desired conversions.

Condensation reactions are typically carried out at temperatures nearroom temperature, although for non-equilibrating, kinetically controlledcondensations reduced temperatures (0° C. to −100° C.) are also common.Solvents can be either protic (common in equilibrating reactions) oraprotic (common in kinetically controlled reactions). Standard synthetictechniques such as azeotropic removal of reaction by-products and use ofanhydrous reaction conditions (e.g. inert gas environments) are commonin the art and will be applied when applicable.

Protecting Groups. The term “protecting group”, “blocking group”, or“PG” refers to any group which, when bound to a hydroxy or otherheteroatom prevents undesired reactions from occurring at this group andwhich can be removed by conventional chemical or enzymatic steps toreestablish the hydroxyl group. The particular removable blocking groupemployed is not always critical and preferred removable hydroxylblocking groups include conventional substituents such as, for example,allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidene, phenacyl,methyl methoxy, silyl ethers (e.g., trimethylsilyl (TMS),t-butyl-diphenylsilyl (TBDPS), or t-butyldimethylsilyl (TBS)) and anyother group that can be introduced chemically onto a hydroxylfunctionality and later selectively removed either by chemical orenzymatic methods in mild conditions compatible with the nature of theproduct. The R groups of Formula (I) can also be protecting groups, asdescribed herein.

Suitable hydroxyl protecting groups are known to those skilled in theart and disclosed in more detail in T. W. Greene, Protecting Groups InOrganic Synthesis; Wiley: New York, 1981 (“Greene”) and the referencescited therein, and Kocienski, Philip J.; Protecting Groups (Georg ThiemeVerlag Stuttgart, New York, 1994), both of which are incorporated hereinby reference.

Protecting groups are available, commonly known and used, and areoptionally used to prevent side reactions with the protected groupduring synthetic procedures, i.e. routes or methods to prepare thecompounds by the methods of the invention. For the most part thedecision as to which groups to protect, when to do so, and the nature ofthe chemical protecting group “PG” will be dependent upon the chemistryof the reaction to be protected against (e.g., acidic, basic, oxidative,reductive or other conditions) and the intended direction of thesynthesis.

Protecting groups do not need to be, and generally are not, the same ifthe compound is substituted with multiple PGs. In general, PG will beused to protect functional groups such as carboxyl, hydroxyl, thio, oramino groups and to thus prevent side reactions or to otherwisefacilitate the synthetic efficiency. The order of deprotection to yieldfree, deprotected groups is dependent upon the intended direction of thesynthesis and the reaction conditions to be encountered, and may occurin any order as determined by the artisan.

Various functional groups of the compounds of the invention may beprotected. For example, protecting groups for —OH groups (whetherhydroxyl, carboxylic acid, or other functions) include “ether- orester-forming groups”. Ether- or ester-forming groups are capable offunctioning as chemical protecting groups in the synthetic schemes setforth herein. However, some hydroxyl and thio protecting groups areneither ether- nor ester-forming groups, as will be understood by thoseskilled in the art. For further detail regarding carboxylic acidprotecting groups and other protecting groups for acids, see Greene,cited above. Such groups include by way of example and not limitation,esters, amides, hydrazides, and the like.

Salts and Solvates

Pharmaceutically acceptable salts of compounds described herein arewithin the scope of the invention and include acid or base additionsalts which retain the desired pharmacological activity and are notbiologically undesirable (e.g., the salt is not unduly toxic,allergenic, or irritating, and is bioavailable). When a compound has abasic group, such as, for example, an amino group, pharmaceuticallyacceptable salts can be formed with inorganic acids (such ashydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, andphosphoric acid), organic acids (e.g. alginate, formic acid, aceticacid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaricacid, lactic acid, maleic acid, citric acid, succinic acid, malic acid,methanesulfonic acid, benzenesulfonic acid, naphthalene sulfonic acid,and p-toluenesulfonic acid) or acidic amino acids (such as aspartic acidand glutamic acid). When the compound of the invention has an acidicgroup, such as for example, a carboxylic acid group, it can form saltswith metals, such as alkali and earth alkali metals (e.g. Na⁺, Li⁺, K⁺,Ca²⁺, Mg²⁺, Zn²⁺), ammonia or organic amines (e.g. dicyclohexylamine,trimethylamine, triethylamine, pyridine, picoline, ethanolamine,diethanolamine, triethanolamine) or basic amino acids (e.g. arginine,lysine and ornithine). Such salts can be prepared in situ duringisolation and purification of the compounds or by separately reactingthe purified compound in its free base or free acid form with a suitableacid or base, respectively, and isolating the salt thus formed.

Many of the molecules disclosed herein contain one or more ionizablegroups [groups from which a proton can be removed (e.g., —COOH) or added(e.g., amines) or which can be quaternized (e.g., amines)]. All possibleionic forms of such molecules and salts thereof are intended to beincluded individually in the disclosure herein. With regard to salts ofthe compounds described herein, one of ordinary skill in the art canselect from among a wide variety of available counterions those that areappropriate for preparation of salts of this invention for a givenapplication. In specific applications, the selection of a given anion orcation for preparation of a salt may result in increased or decreasedsolubility of that salt.

Examples of suitable salts of the compounds described herein includetheir hydrochlorides, hydrobromides, sulfates, methanesulfonates,nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g.,(+)-tartrates, (−)-tartrates or mixtures thereof including racemicmixtures), succinates, benzoates and salts with amino acids such asglutamic acid. These salts may be prepared by methods known to thoseskilled in the art. Also included are base addition salts such assodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the present invention containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of acceptable acid addition salts include those derived frominorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived organicacids like acetic, propionic, isobutyric, maleic, malonic, benzoic,succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike. Certain specific compounds of the invention can contain both basicand acidic functionalities that allow the compounds to be converted intoeither base or acid addition salts.

Certain compounds of the invention can exist in unsolvated forms as wellas solvated forms, including hydrated forms. In general, the solvatedforms are equivalent to unsolvated forms and are encompassed within thescope of the invention. Certain compounds of the invention may exist inmultiple crystalline or amorphous forms. In general, all physical formsare equivalent for the uses contemplated by the invention and areintended to be within the scope of the invention.

The term “solvate” refers to a solid compound that has one or moresolvent molecules associated with its solid structure. Solvates can formwhen a compound is crystallized from a solvent. A solvate forms when oneor more solvent molecules become an integral part of the solidcrystalline matrix upon solidification. The compounds of the formulasdescribed herein can be solvates, for example, ethanol solvates. Anothertype of a solvate is a hydrate. A “hydrate” likewise refers to a solidcompound that has one or more water molecules intimately associated withits solid or crystalline structure at the molecular level. Hydrates canform when a compound is solidified or crystallized in water, where oneor more water molecules become an integral part of the solid crystallinematrix. The compounds of the formulas described herein can be hydrates.

Pharmaceutical Compositions

The following describes information relevant to pharmaceutical andpharmacological embodiments and is further supplemented by informationin the art available to one of ordinary skill. The exact formulation,route of administration and dosage can be chosen by an individualphysician or clinician in view of a patient's condition (see e.g., Finglet al., in The Pharmacological Basis of Therapeutics, 1975, Ch. 1).

It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicity,or to organ dysfunctions, etc. Conversely, the attending physician wouldalso know to adjust treatment to higher levels if the clinical responsewere not adequate (in light of or precluding toxicity aspects). Themagnitude of an administered dose in the management of the disorder ofinterest can vary with the severity of the condition to be treated andto the route of administration. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency, can also varyaccording to circumstances, e.g. the age, body weight, and response ofthe individual patient. A program comparable to that discussed abovealso may be used in veterinary medicine.

Depending on the specific conditions being treated and the targetingmethod selected, such agents may be formulated and administeredsystemically or locally. Techniques for formulation and administrationmay be found in Alfonso and Gennaro (1995) and elsewhere in the art.

The compounds can be administered to a patient in combination with apharmaceutically acceptable carrier, diluent, or excipient. The phrase“pharmaceutically acceptable” refers to those ligands, materials,compositions, and/or dosage forms that are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, diluents, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts, buffers,preservatives, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the chemotherapeutic or pharmaceutical compositions is contemplated.

A DNQ_(d) or DNQ compound may be combined with different types ofcarriers depending on whether it is to be administered in solid, liquidor aerosol form, and whether it need to be sterile for such routes ofadministration as injection. The present invention can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, intraperitoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, topically, locally, injection, infusion,continuous infusion, localized perfusion bathing target cells directly,via a catheter, via a lavage, in lipid compositions (e.g., liposomes),or by other method or any combination of the forgoing as would be knownto one of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,incorporated herein by reference).

The actual dosage amount of a composition of the present inventionadministered to a patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

When administered to a subject, effective amounts will depend, ofcourse, on the particular cancer being treated; the genotype of thespecific cancer; the severity of the cancer; individual patientparameters including age, physical condition, size and weight,concurrent treatment, frequency of treatment, and the mode ofadministration. These factors are well known to the physician and can beaddressed with no more than routine experimentation. In someembodiments, it is preferred to use the highest safe dose according tosound medical judgment.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of a DNQ_(d) or DNQ compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 0.1 mg/kg/body weight, 0.5mg/kg/body weight, 1 mg/kg/body weight, about 5 mg/kg/body weight, about10 mg/kg/body weight, about 20 mg/kg/body weight, about 30 mg/kg/bodyweight, about 40 mg/kg/body weight, about 50 mg/kg/body weight, about 75mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/bodyweight, about 350 mg/kg/body weight, about 500 mg/kg/body weight, about750 mg/kg/body weight, to about 1000 mg/kg/body weight or more peradministration, and any range derivable therein. In non-limitingexamples of a derivable range from the numbers listed herein, a range ofabout 10 mg/kg/body weight to about 100 mg/kg/body weight, etc., can beadministered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including, but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

Actives described herein such as DNQ_(d) or DNQ compounds may beformulated into a composition in a free base, neutral or salt form.Pharmaceutically acceptable salts include the salts formed with the freecarboxyl groups derived from inorganic bases such as for example,sodium, potassium, ammonium, calcium or ferric hydroxides; or suchorganic bases as isopropylamine, triethylamine, histidine or procaine.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are optionally provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used, which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising, but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose (HPC); or combinations thereof suchmethods. In many cases, it will be preferable to include isotonicagents, such as, for example, sugars, sodium chloride or combinationsthereof.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount of the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and/or the other ingredients. Inthe case of sterile powders for the preparation of sterile injectablesolutions, suspensions or emulsion, the preferred methods of preparationare vacuum-drying or freeze-drying techniques which yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered liquid medium thereof. The liquid mediumshould be suitably buffered if necessary and the liquid diluent firstrendered isotonic prior to injection with sufficient saline or glucose.

The composition should be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Thus, preferred compositionshave a pH greater than about 5, preferably from about 5 to about 8, morepreferably from about 5 to about 7. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

Formulation of DNQ Compounds for in vivo Administration

The aqueous solubility of DNQ at pH 7.4 in phosphate buffered saline(PBS) was measured by LC-MS. DNQ was sonicated for 30 minutes in PBSthen undissolved solid was removed by filtration through a 0.45 μmsyringe filter and the filtrate was analyzed by LC-MS (λ=275 nm, ESI-TOFin negative mode). The optimal sonication time was determined bysonicating DNQ for 1, 5, 10, and 30 minutes. While the concentration ofDNQ in solution increased substantially between 1, 5, and 10 minutes,there was only a minor difference between 10 and 30 minutes. During the30 minute sonication the water bath warmed to 45° C. (samples werecooled to room temperature before filtration). A calibration curve wasgenerated from 1-100 μM by dissolving DNQ in methanol to a concentrationof 500 μM and making dilutions of this stock in 80:20 water:methanol.The calibration curve (measure by UV absorbance) was linear over thisrange; 1 μM was approximately the limit of detection. The solubility ofDNQ in PBS was measured to be 115 μM. The solution was very pale yellow.

Because of the poor aqueous solubility of DNQ we investigated the use of2-hydroxypropyl-beta-cyclodextrin (HPβCD), a common excipient, toimprove the solubility of DNQ. In the absence of HPβCD, the solubilityof DNQ increases significantly in strongly basic solutions and DNQprecipitates when the pH is returned to neutral. However, in thepresence of a sufficient amount of HPβCD, DNQ does not precipitate whenthe pH is returned to neutral. This same neutral solution of DNQ inHPβCD cannot be made directly (i.e. without pH adjustment). Thisindicates that DNQ compounds deprotonate in base and this deprotonatedmolecule forms a tight complex with HPβCD which is stable enough toprevent protonation as the pH decreases. The only proton on DNQ thatmight reasonably be deprotonated in aqueous base is the N—H. Althoughthe acidity of the N—H bond of DNQ has not been measured, it has beenmeasured for a derivative of DNQ and found to have a pKa of 8.0.

The protocol for formulating DNQ compounds in HPβCD is as follows: theDNQ compound is slurried in a 20% solution of HPβCD in pH 7.4 PBS andthe pH is then increased by the addition of 10 M NaOH to inducedissolution of the DNQ compound. The pH is returned to pH 7.5-8.0 by thecareful addition of 1 M HCl. A 3.3 mM solution of the DNQ compound canbe made by this method which is stable at least 24 hours. Thisrepresents a 30-fold increase in solubility of DNQ over PBS alone. Weinitially chose a 20% HPβCD solution. However, we have found that β-lapwas formulated as a 40% solution of HPβCD for human clinical trials andour experience with DNQ indicates that the concentration of DNQincreases linearly with that of HPβCD; thus a 40% HPβCD solution wouldpermit the creation of a 6.6 mM solution of DNQ and other DNQ compounds.

Combination Therapy

Active ingredients described herein (e.g., compounds of Formula (I)) canalso be used in combination with other active ingredients. Suchcombinations are selected based on the condition to be treated,cross-reactivities of ingredients and pharmaco-properties of thecombination. For example, when treating cancer, the compositions can becombined with other anti-cancer compounds (such as paclitaxel orrapamycin).

It is also possible to combine a compound of the invention with one ormore other active ingredients in a unitary dosage form for simultaneousor sequential administration to a patient. The combination therapy maybe administered as a simultaneous or sequential regimen. Whenadministered sequentially, the combination may be administered in two ormore administrations.

The combination therapy may provide “synergy” and “synergistic”, i.e.the effect achieved when the active ingredients used together is greaterthan the sum of the effects that results from using the compoundsseparately. A synergistic effect may be attained when the activeingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined formulation; (2) delivered by alternationor in parallel as separate formulations; or (3) by some other regimen.When delivered in alternation therapy, a synergistic effect may beattained when the compounds are administered or delivered sequentially,e.g. in separate tablets, pills or capsules, or by different injectionsin separate syringes. In general, during alternation therapy, aneffective dosage of each active ingredient is administered sequentially,i.e. serially, whereas in combination therapy, effective dosages of twoor more active ingredients are administered together. A synergisticanti-cancer effect denotes an anti-cancer effect that is greater thanthe predicted purely additive effects of the individual compounds of thecombination.

Combination therapy is further described by U.S. Pat. No. 6,833,373(McKearn et al.), which includes additional active agents that can becombined with the compounds described herein, and additional types ofcancer and other conditions that can be treated with a compounddescribed herein.

Accordingly, it is an aspect of this invention that a DNQ_(d) or DNQ canbe used in combination with another agent or therapy method, preferablyanother cancer treatment. A DNQ_(d) or DNQ may precede or follow theother agent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and expression construct are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not elapse between the time of each delivery, suchthat the agent and expression construct would still be able to exert anadvantageously combined effect on the cell. For example, in suchinstances, it is contemplated that one may contact the cell, tissue ororganism with two, three, four or more modalities substantiallysimultaneously (i.e., within less than about a minute) with the activeagent(s). In other aspects, one or more agents may be administeredwithin about 1 minute, about 5 minutes, about 10 minutes, about 20minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about9 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours,about 24 hours, about 28 hours, about 31 hours, about 35 hours, about 38hours, about 42 hours, about 45 hours, to about 48 hours or more priorto and/or after administering the active agent(s). In certain otherembodiments, an agent may be administered within from about 1 day, about2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 8days, about 9 days, about 12 days, about 15 days, about 16 days, about18 days, about 20 days, to about 21 days prior to and/or afteradministering the active agent(s). In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several weeks (e.g., about 1, about 2, about 3, about 4,about 6, or about 8 weeks or more) lapse between the respectiveadministrations.

Administration of the chemotherapeutic compositions of the presentinvention to a patient will follow general protocols for theadministration of chemotherapeutics, taking into account the toxicity,if any. It is expected that the treatment cycles would be repeated asnecessary. It also is contemplated that various standard therapies oradjunct cancer therapies, as well as surgical intervention, may beapplied in combination with the described active agent(s). Thesetherapies include but are not limited to chemotherapy, radiotherapy,immunotherapy, gene therapy and surgery.

Chemotherapy

Cancer therapies can also include a variety of combination therapieswith both chemical and radiation based treatments. Combinationchemotherapies include the use of chemotherapeutic agents such as,cisplatin, etoposide, irinotecan, camptostar, topotecan, paclitaxel,docetaxel, epothilones, taxotere, tamoxifen, 5-fluorouracil,methoxtrexate, temozolomide, cyclophosphamide, SCH 66336, R115777,L778,123, BMS 214662, IRESSA™ (gefitinib), TARCEVA™ (erlotinibhydrochloride), antibodies to EGFR, GLEEVEC™ (imatinib), intron, ara-C,adriamycin, cytoxan, gemcitabine, uracil mustard, chlormethine,ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine,triethylenethiophosphoramine, busulfan, carmustine, lomustine,streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine,6-thioguanine, fludarabine phosphate, pentostatine, vinblastine,vincristine, vindesine, bleomycin, doxorubicin, dactinomycin,daunorubicin, epirubicin, idarubicin, mithramycin, deoxycoformycin,Mitomycin-C, L-Asparaginase, teniposide, 17α-Ethinylestradiol,Diethylstilbestrol, testosterone, prednisone, fluoxymesterone,dromostanolone propionate, testolactone, megestrolacetate,methylprednisolone, methyltestosterone, prednisolone, triamcinolone,chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine,medroxyprogesterone acetate, leuprolide, flutamide, toremifene,goserelin, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane,mitoxantrone, levamisole, navelbene, anastrazole, letrazole,capecitabine, reloxafine, droloxafine, hexamethylmelamine, Avastin,herceptin, Bexxar, Velcade, Zevalin, Trisenox, Xeloda, Vinorelbine,Porfimer, Erbitux™ (cetuximab), Liposomal, Thiotepa, Altretamine,Melphalan, Trastuzumab, Lerozole, Fulvestrant, Exemestane, Fulvestrant,Ifosfomide, Rituximab, C225, Campath, carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16),tamoxifen, raloxifene, estrogen receptor binding agents, paclitaxel,gemcitabine, navelbine, farnesyl-protein transferase inhibitors,transplatinum, 5-fluorouracil, vincristine, vinblastine andmethotrexate, or any analog or derivative variant of the foregoing.

Radiotherapy.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as .gamma.-rays, X-rays, and/or thedirected delivery of radioisotopes to tumor cells. Other forms of DNAdamaging factors are also contemplated such as microwaves andUV-irradiation. It is most likely that all of these factors affect abroad range of damage on DNA, on the precursors of DNA, on thereplication and repair of DNA, and on the assembly and maintenance ofchromosomes. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (e.g., 3 to 4 wks), tosingle doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopesvary widely, and depend on the half-life of the isotope, the strengthand type of radiation emitted, and the uptake by the neoplastic cells.The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

Immunotherapy.

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually affect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionucleotide, ricin A chain, cholera toxin, pertussis toxin, etc.)and serve merely as a targeting agent. Alternatively, the effector maybe a lymphocyte carrying a surface molecule that interacts, eitherdirectly or indirectly, with a tumor cell target. Various effector cellsinclude cytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, inconjunction with gene therapy. The general approach for combined therapyis discussed below. Generally, the tumor cell must bear some marker thatis amenable to targeting, i.e., is not present on the majority of othercells. Many tumor markers exist and any of these may be suitable fortargeting in the context of the present invention. Common tumor markersinclude carcinoembryonic antigen, prostate specific antigen, urinarytumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72,HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155.

Gene Therapy.

In yet another embodiment, the secondary treatment is a secondary genetherapy in which a therapeutic polynucleotide is administered before,after, or at the same time a first chemotherapeutic agent. Delivery ofthe chemotherapeutic agent in conjunction with a vector encoding a geneproduct will have a combined anti-hyperproliferative effect on targettissues.

Surgery.

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies. Curative surgeryincludes resection in which all or part of cancerous tissue isphysically removed, excised, and/or destroyed. Tumor resection refers tophysical removal of at least part of a tumor. In addition to tumorresection, treatment by surgery includes laser surgery, cryosurgery,electrosurgery, and microscopically controlled surgery (Mohs' surgery).It is further contemplated that the present invention may be used inconjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

One of ordinary skill in the art will appreciate that startingmaterials, biological materials, reagents, synthetic methods,purification methods, analytical methods, assay methods, and biologicalmethods other than those specifically exemplified can be employed in thepractice of the invention without resort to undue experimentation. Allart-known functional equivalents, of any such materials and methods areintended to be included in this invention. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention. The invention may be further understood bythe following non-limiting examples.

Abbreviations used in the Schemes and Examples may include thefollowing:

-   A549=adenocarcinomic human alveolar basal epithelial cells-   ATP=adenosine triphosphate-   β-lap=β-lapachone-   DHE=dihydroethidium-   DNQ=Deoxynyboquinone-   DNQ_(d)=Any analog or derivative of deoxynyboquinone-   ELISA=enzyme-linked immunosorbent assay-   h=hour(s)-   H596=[NCl—H596] human lung adenosquamous carcinoma cell line-   HT1080=primate fibrosarcoma cell line-   LD₅₀=lethal dose having 50% probability of causing death-   LD₉₀=lethal dose having 90% probability of causing death-   LD₁₀₀=lethal dose having 100% probability of causing death-   MCF-7=human breast adenocarcinoma cell line-   MDA-MB-231=human breast cancer cell line-   MIA-PaCa2=Pancreatic cancer cell line-   mins=minute(s)-   NADH=nicotinamide adenine dinucleotide-   NQO1=NAD(P)H:quinone oxidoreductase 1-   NSCLC=non-small-cell lung cancer cells-   OCR=oxygen consumption rates-   p53=a tumor suppressor protein-   PC-3=human prostate cancer cell line-   ROS=reactive oxygen species-   ±SE=standard error-   siRNA=small interfering ribonucleic acid-   shRNA=small hairpin ribonucleic acid-   μM=micromolar-   nM=nanomolar-   μmol=micromole

EXAMPLES Example 1 Base Excision Repair Inhibition SynergisticallyEnhances β-Lapachone-Mediated Cell Death for Tumor-Selective Therapy ofPancreatic Cancer

Blocking DNA base excision (BER) or SSB repair processes with PARP1inhibitors can dramatically increase efficacy, and significantly lowerrequired doses, of NQO1 bioactivatable drugs against NQO1+over-expressing cancers such as pancreatic cancers. To demonstrate thesemethods, experiments were performed to mechanistically show thatinhibiting BER or SSB repair synergizes with NQO1 bioactivatable drugsagainst NQO1+ vs. NQO1− or shRNA knockdown pancreatic cancer cells invitro. Additionally, experiments were performed to optimize MeOX orPARP1 inhibitors to enhance NQO1 bioactivatable drug efficacy in vivo.The combination therapy can thus be used as a tumor-specific approachusing DNA repair inhibitors (e.g., BER and PARP1 inhibitors).

This example demonstrates the tumor-specific efficacy of NQO1bioactivatable drugs against pancreatic, as well as otherNQO1-overexpressing cancers; and demonstrates ‘tumor-specificity’ forPARP1 inhibitors outside current, limited, ‘synthetic lethal’ approachesfor cancer therapy.

The synergistic effects of adding methoxyamine (MeOX) with β-lap againstNQO1 over-expressing pancreatic cancer cells were examined. MeOX+β-lapsynergy resulted in: a, enhanced lethality of sub-lethal doses of β-lap,reducing the shoulder (Dq), increasing the lethality rate (Do), andinducing apoptosis (TUNEL+) in NQO1+, but not in NQO1−, MIA PaCa-2pancreatic cells; b, increased DNA lesion formation only in tumor cells;c, dramatic losses in ATP levels, with little recovery; and d, dramaticsuppression of glycolysis. Thus, MeOX enhances PARP1 hyperactivation andsynergistic cell killing by β-lap. Similar results were noted inshRNA-XRCC1 knockdown cells. However, Ogg1 knockdown cells were renderedresistant ß-lap. Mechanistically, the data indicate that PARP1 detectsMeOX-AP modified sites or SSBs, allowing PARP1 hyperactivation andsynergistic cell death. Because MeOX is a nontoxic agent, thecombination of agents can provide therapies for the treatment ofpancreatic, as well as other NQO1 overexpressing solid cancers.

FIG. 1 shows the expression of NQO1 and Catalase in pancreatic tumor vs.associated normal tissue. Catalase is thus significantly over-expressionin normal vs. tumor tissue. NQO1:Catalase ratios in pancreatic cancervs. normal tissue are a major determinant of efficacy for NQO1bioactivatable drugs such ß-lap and DNQ (FIG. 2). Particularly in thepresence of a high NQO1:Catalase ratio, NQO1 bioactivatable drugs killby tumor-specific DNA damage, inducing PARP1 hyperactivation and uniqueprogrammed necrotic cell death. Tumor-specific lethality is independentof p52 status, Bax/bak loss, oncogene activation status, cell cyclestatus, and hypoxia.

NQO1 is a principal determinant of ß-lap cytotoxicity, and functionalinhibition of NQO1 by shRNA-NQO1 knockdown protects from cell deathafter β-lap exposures (FIG. 3). ß-Lap-induced DNA damage isNQO1-dependent, tumor-specific, and is significantly decreased by NQO1shRNA-knockdown (FIG. 4). Also, NQO1 bioactivatable drugs cause dramaticsuppression of glycolysis and loss of ATP that can inhibit DNA repair(FIG. 5).

Altering BER or SSB repair can enhance NQO1 bioactivatable drug efficacyin a tumor-selective manner. siRNA-mediated knockdown of the Ogg1glycosylase renders s-lap-treated pancreatic cancer cells resistant toß-lapachone (FIG. 6), however NQO1 bioactivatable drugs cause SSBs andbase lesions in a tumor-specific manner, thus they can be used to makeDNA repair inhibitors tumor-selective (FIG. 7). PARP1 inhibitorssynergize with NQO1 bioactivatable drugs for enhanced efficacy in NQO1+cancer cells, such as pancreatic cancer cells (FIG. 8). Also, NQO1bioactivatable lethality is enhanced by MeOX, with accompanyingmetabolic effects (FIG. 9). Furthermore, β-Lapachone has significantantitumor efficacy against MIA PaCa-2 tumor xenografts, as illustratedby the preclinical study data shown in FIG. 10.

Accordingly, this examples and its supporting data show that NQO1bioactivatable drugs induce DNA base damage and single strand DNAbreaks, and that inhibiting Base Excision Repair (BER) enhancesß-lap-mediated pancreas cancer-selective lethality. Additionally,inhibiting PARP1 activity enhances ß-lap efficacy against NQO1+ pancreascancers. Finally, use of NQO1 bioactivatable drugs, such as compounds ofthe formulas of FIG. 11 and the specific compounds of FIG. 12, providesfor tumor-selective use of DNA repair inhibitors, and NQO1bioactivatable drugs cause dramatic effects such as the suppression ofglucose metabolism.

Example 2 DNQ Compound and β-Lapachone Data and Therapy

IB-DNQ (DNQ-87; FIG. 12) works at much lower doses versus ß-lapachoneand at doses equivalent to the parental DNQ compound. As shown in FIG.13, it is effective against breast cancer cells in an NQO1-dependentmanner, as well as triple-negative breast cancer cells. Unlikeβ-lapachone (ß-lap), efficacy of DNQ87 increases in an NQO1-dependentmanner and the therapeutic window is larger (FIG. 14). As shown in FIG.15, DNQ87 causes cell death that can be blocked by dicoumarol, catalase,and BAPTA-AM (a calcium chelator) (A), in descending order andconsistent with the proposed pathway of cell death caused by NQO1bioactivatable drugs (B). (C) PARP1 hyperactivation caused by DNQ87exposure measured by PAR-PARP1 formation, highlighted byμ-calpain-mediated p53 cleavage (C) and atypical cleavage of PARP1 to^(˜)60 kDa proteolytic fragments during cell death (D). DNQ87 alsocauses DNA lesions (DNA double strand breaks) in a delayed manner,monitored by gamma-H2AX, phosphorylation of ATM at ser1981, andphosphorylation of DNA-PKcs at site Thr1892 (FIG. 16). These data alsoindicate that DSB repair inhibitors could also be used to enhance DNQ87lethality. Importantly alkaline elution shows extensive DNA base lesionsand DNA single strand breaks, whereas the same cells assessed by neutralcomet assays shows no DNA lesions.

DSB formation, monitored by P-ATM and P-H2AX are delayed in NQO1overexpressing human breast cancer cells, and only occur after PARP1hyperactivation. IB-DNQ (DNQ-87; FIG. 12) causes massive H₂O₂ formationleading to DNA base and single strand breaks that are quickly recognizedby PARP1, which protects the DNA and stimulates base excision and DNAsingle strand break repair. Only when PARP1 is exhausted through itshyperactivation are DNA double strand breaks noted (see FIG. 17). FIG.18 shows the therapeutic window of DNQ87 using dicoumarol to mimicnormal tissue in NQO1 overexpressing MCF-7 human breast cancer cells. Asillustrated by FIG. 19, exposure to NQO1 bioactivatable drugs causesextensive 8-oxoguanine levels, levels equivalent to 500 μM H₂O₂exposures. Knockdown of the glycosylase, Ogg1, which preferentiallydetects 8-oxo-guanine (8-OG), results in dramatic resistance to NQO1bioactivatable drugs, such as ß-lapachone. A) ß-lapachone exposurecauses extensive formation of 8-oxoguanine. B, C) Two separateexperiments are shown. Mia-Paca2 pancreatic cancer cells were exposed tosiRNA-scrambled or siRNA-specific for Ogg1 for 24 hours, then cells weretreated with ß-lapachone for 2 hours at the indicated doses. Survival,measured by colony forming ability assays were then performed andgraphed with ß-lapachone doses used.

NQO1 expressing cells create high levels of H₂O₂ that are not scavengedby catalase, due to its lowered levels in cancer cells. In contrast,normal tissues have low NQO1 levels and if H₂O₂ is created by exposureto the drug, elevated levels of Catalase scavenges the obligate ROS forthis agent. Normal tissues are therefore protected (see FIG. 2).Knowledge of DNA damage created by DNQ, ß-lapachone or their respectiveanalogs allows new and non-obvious strategies for enhanced lethality.Conversely, DNA repair inhibitors fail commonly because they lacktumor-selectively. The NQO1-dependent DNA lesions created exclusively inspecific solid tumors, results in specific DNA base damage (e.g.,8-oxyguanine), which through futile DNA repair processes results inPARP1 hyperactivation. Knowledge of this damage results in two separatestrategies to enhance the tumor-specific lethalities of NQO1bioactivatable drugs: (A) using DNA apurinic/apyrimidinic (AP site)modifying agents, such as methoxyamine (MeOX), which is currently inclinical trials; and (B) use of PARP1 inhibitors, which prevents itshyperactivation, but also prevents repair of DNA single strand and thenDNA double strand breaks caused by futile BER repair and DNA replicationin cancer cells. Since we previously have obtained data showing thatPARP1 hyperactivation is required for lethality by NQO1 bioactivatabledrugs, inhibiting PARP1 activity to achieve synergy is a surprisingresult. Cells die by enhanced PARP1 hyperactivation and programmed celldeath by the mechanism in (A) using MeOX, while cells die by normalapoptosis in the strategy outlined in B using PARP1 inhibitors (see FIG.7).

As shown in FIG. 20, methoxyamine enhances ß-lapachone-inducedlethality. A, Addition of nonlethal doses of methoxyamine (MX or MeOX)to cells exposed to ß-lapachone results in synergistic lethality of MiaPaca2 pancreatic cancer cells. B and C, Methoxyamine (MeOX) is anAP-site modifying chemical that is nontoxic to >100 mM as measured byrelative survival and ATP loss. Optimal concentrations in combinationwith NQO1 bioactivatable drugs are between 6-12 mM. FIG. 21 illustratesProof of Principle studies. A. Methoxyamine (MeOX) augments ß-lapachoneß-lap)-induced lethality in MiaPaca2 pancreatic cancer cells. B.Addition of MeOX modifies AP sites and therefore decreases signal of APsite formation in ß-lap+MeOX exposed cells. MeOX dose was 12 mM, ß-lapDose was 6 μM. C,D. XRCC1 is a scaffolding protein that enables baseexcision repair (BER). Elimination of XRCC1 causes elevated AP sites andDNA single strand breaks, at which PARP1 can bind and becomeactivated/hyperactivated. Knockdown of XRCC1 (C) enhances the lethalityof ß-lapachone treatment.

As shown in FIG. 22, addition of nontoxic doses of methoxyamine (MeOX)greatly enhances DNA damage in Mia PaCa-2 cells exposed to sublethaldoses ß-lapachone (2 μM). A) DNA double strand breaks (DSBs), monitoredby gamma-H₂AX, are greatly enhanced by the addition of nontoxic doses ofmethoxyamine (MeOX). MeOX, 12 mM; Dicoumarol, NQO1 inhibitor, 50 μM;ß-lapachone doses, indicated or 2 μM. For Mia Paca2 cells, 2-2.5 μM issublethal, 6 μM is lethal. H₂O₂ dose was 500 μM, 2 h. All treatmentswere 2 h in duration. Cells were treated 2× with MeOX, as a 2 hpretreatment and then for 2 h in combination with ß-lap whereapplicable.

Methoxyamine pre- and co-treatments prevent ATP recovery responses inMia PaCa-2 pancreatic cancer cells exposed to NQO1 bioactivatable drugs,such as ß-lapachone (ß-lap) (FIG. 23). A, ATP recovery responses areprevented by MeOX and XRCC1 knockdown, presumably by enhanced PARP1hyperactivation. B, MeOX addition to ß-lap-treated Mia PaCa2 cellsprevents ATP recovery. C, BAPTA-AM, a calcium chelator, prevents MeOXsynergistic ATP loss. BAPTA-AM was used at 6 μM. D, Addition of MeOXenhances NAD+ loss, consistent with enhanced PARP1 hyperactivation.

Addition of Methoxyamine (MeOX) prevents ATP recovery responses inß-lap-treated Mia PaCa-2 cells at lethal and sublethal doses (FIG. 24).Lethal doses of ß-lapachone were 6 and 4 μM, whereas 3.0 and 2.5 μM aresublethal doses of ß-lapachone (ß-lap).

Methyl pyruvate (MP) suppresses β-lapachone-induced cell death (FIG.25). While the effects could be due to recovery of the TCA cycle, MP isalso an outstanding oxygen free radical (reactive oxygen species, ROS)scavenger.

Addition of Methoxyamine attenuates MP effects, presumably because lessinitial ROS (H₂O₂) is required to create unrepairable (andMeOX-modified) AP sites (FIG. 26). ß-Lap doses indicated, MP used at 1or 5 mM, and MeOX was used at 12 mM.

Methoxyamine (MeOX) accelerates PARP1 hyperactivation-induced NAD+/ATPloss, further accelerating reparation in ß-lapachone-exposed Mia PaCa-2cells (FIG. 27). A. ß-Lapachone enhances oxygen consumption rates (OCR)in Mia PaCa2 cells. Lethal doses of ß-lap cause dramatic OCR spikes, andsubsequent losses of all metabolic capacity. Sublethal doses of ß-lapcause consistently elevated OCRs over time. Both responses areNQO1-mediated. B, addition of MeOX enhances OCR rates in combinationwith sublethal doses of ß-lap, similar to a lethal dose of ß-lapachone(4 μM), presumably because of the loss of NAD+ and ATP derived fromPARP1 hyperactivation.

NQO1 bioactivatable drug treatment suppresses glycolysis in Mia PaCa2cells (FIG. 28). Both glucose utilization (A) and lactate production (B)are suppressed. A549 NSCLC cells were co-treated with 12 mM Methoxyamine(MeOX) and DNQ 87 for 2 h (FIG. 29) and relative survival was measured(as in Huang et al., Cancer Res., 2012).

PARP1 Inhibition Enhances the Tumor-Selective Lethality of NQO1Bioactivatable Drugs.

PARP1 inhibitors enhance the lethality of NQO1 bioactivatable drugs(FIG. 30). Mia PaCa-2 cells were treated for 2 hours with the inhibitorsnoted in FIG. 30 and then with DNQ+the inhibitors (all at 15 μM) for 2hours. Cells were then washed and allowed to grow for 7 days andrelative survival was assessed as in Huang et al., Cancer Res., 2012,72(12), 3038-3047, which also provides additional useful methods andtechniques that can be incorporated into the method described herein.All of the inhibitors, except BSI-201, inhibited PAR formation. Furtheranalyses showed that BSI-201 was not an efficacious PARP1 inhibitor, butdid cause DNA damage, which is the mechanism by which this inhibitorsynergized with NQO1 bioactivatable drugs (DNQ (shown) or ß-lap). FIG.31 shows data for the determination of nonlethal doses of PARP1inhibitors alone in A549 NSCLC cells. FIG. 32 shows data obtained forthe PARP1 inhibitor, AG014699.

AG014699 enhances the NQO1-dependent lethality of ß-lapachone in A549NSCLC cells (FIG. 33). A, control to demonstrate synthetic lethality ofdrug alone against a BRCA1−/− CAPAN-1 cells, whereas other BRAC1wild-type cancer cells (A549 and Mia PaCa-2 cells are completelyresistant to the PARP1 inhibitor. B, Demonstration that AG014699inhibits PARP1 hyperactivation in response to ß-lap. C, Synergisticlethality of AG014699 in combination with ß-lap, which is prevented withthe NQO1 inhibitor, dicoumarol. D, Dose-response of AG014699 incombination with ß-lap, and reversal by dicoumarol addition.

PARP1 knockdown enhances lethality of ß-lapachone in triple negativeMDA-MB-231 (231) breast cancer cells (FIG. 34). A. Stable PARP1 shRNAknockdown cells developed by us (Bentle et al., JBC 2006). Western blotsdemonstrate stable knockdown in cells expressing or lacking NQO1. B.Demonstrates dramatically reduced PAR-PARP1 formation in PARP1 knockdowncells. Note that DNA double strand breaks occur much sooner than inPARP1 wild-type cells. C. PARP1 knockdown sensitizes cells to ß-lap inlong-term survival assays. Although PARP1 knockdown suppressesprogrammed necrosis induced by ß-lap (Bentle et al., JBC 2006), thelong-term consequences are that without PARP1 cells cannot repair DNAlesions created by NQO1 bioactivatable drugs, such ß-lap, and thoselesions are converted to DSBs that eventually cause cells to die throughregular caspase-mediated pathways. D. Addition of AG014699 to PARP1knockdown 231 cells does not significantly enhance their lethalityinduced by ß-lap. All PARP1 inhibitors have shown similar responses,except for BSI-201.

PARP1 knockdown in MCF-7 breast cancer cells also enhances ß-laplethality (FIG. 35). A, B, PARP1 knockdown in NQO1 over-expressing MCF-7cells greatly enhances ß-lap lethality. Note that NQO1 inhibition bydicoumarol (DIC) prevents synergy by blocking NQO1 bioactivation.

NQO1+ H596 NSCLC cells treated with nontoxic doses of ß-lap+nontoxicdoses of AG014699 show evidence of apoptosis in the form of cleavedcaspase-3 (FIG. 36). STS, staurosporine (1 μm, 1 h) serves as a positivecontrol. The active form of caspase 3 is the lower band on the blot.

PARP1 shRNA stable knockdown enhances lethality (A) but suppresses ATPloss (B) due to the suppression (loss) of PARP1 hyperactivation (FIG.37).

As shown in FIG. 38, ß-Lap-induced ATP loss is prevented with PARP1shRNA stable knockdown or addition of AG014699, even though synergisticlethality occurs between ß-lap and PARP1 loss or inhibition. FIG. 39-42show studies in Mia Paca-2 and some DNQ combined PARP1 inhibitor data.

FIG. 43 shows pharmacokinetic analysis and target valuation of dC₃micelles in vivo. a, blood concentrations of dC3 and β-lap (convertedfrom dC3). Pharmacokinetic parameters (e.g., t_(1/2)) were calculatedusing a two-compartment pharmacokinetic model. b, Tumor concentrationsof dC3 and β-lap (converted from dC3). FIG. 44 shows DNQ derivativePAR-PARP1 formation in vivo. The MDT pilot study data is shown below inTable 2-1.

TABLE 2-1 DNQ Derivatives Pilot Study of Maximum Tolerated Dose (MTD).Compound NOD/SCID Doses(mg/kg) Dead mice(5 IV.) MTD* DNQ 3 7.5 2 6  87 314 0 ~16 107 3 10 1 8  9-251 3 20 0 20 10-41 3 15 3 7 NOD/SCID: 1million 3LL-Luc cells were injected into each mouse, and after two daysIV was begun. *Pilot experiments indicate a new practical MTD inNOD/SCID, iv, 1x every other day, 5 injections.

A459 NSCLC cells were pretreated with 15 μM AG014699 for 2 hours, thenexposed to the same concentration of AG014699+various concentrations ofDNQ87 for 2 hours as indicated (FIG. 45). Cells were then washed free ofdrug and relative survival performed as previously described (Huang etal., Cancer Res., 2012). Cells treated in this manner by nontoxic dosesof ß-lap show enhanced DNA lesion formation, equivalent to DNA lesionsformed by a lethal dose of ß-lap (8 μM, 2 hours) (FIG. 46).

Catalase Ratio Calculations. FIG. 1 shows NQO1/Catalase levels inmatched (n=59) (A, B), as well as batched human pancreatic tumor vs.associated normal tissues. Importantly, NQO1 is required to‘bioactivate’ DNQ and ß-lapachone and their analogs that are substratesfor the enzyme, whereas Catalase is the one known resistance freeradical scavenging enzyme that can protect against these drugs. Notethat NQO1 ratios are very high in tumor tissue, yet low in associatednormal tissue (E, H). FIG. 47 shows that NQO1/Catalase ratios areelevated in breast cancer tumors, but low in associated normal tissue(C, F). Triple-negative (ER−, PR− and HR−) human breast cancer cells arealso elevated in NQO1 levels compared to associated normal tissue. FIG.48 shows that NQO1 levels are also elevated in non-small cell lungcancers (NSCLCs) versus associated normal tissue (n=105) (A), whereasCatalase is elevated in Normal tissue and lower levels are found inNSCLC tumors (B). Similar to pancreatic and breast cancer versus normaltissue, the NQO1/Catalase ratios are elevated in tumor and lower levelsfound in normal tissue. Note that NQO1 levels are also elevated inprostate cancers (Dong et al., Cancer Res., 2010), although catalaselevels were not assessed in that particular study. The data of FIG. 49provides validation in human NSCLC patient samples of the NQO1/Catalaseratios in tumor versus normal tissue.

Example 3 Pharmaceutical Dosage Forms

The following formulations illustrate representative pharmaceuticaldosage forms that may be used for the therapeutic or prophylacticadministration of a compound of a formula described herein (e.g., DNQ ora DNQ compound, or β-Lap or a β-Lap derivative), a compound specificallydisclosed herein, a pharmaceutically acceptable salt or solvate thereof,or a combination of compounds described herein (hereinafter referred toas ‘Compound X’):

mg/tablet (i) Tablet 1 ‘Compound X’ 10.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 210.0 (ii) Tablet 2 ‘Compound X’ 20.0 Microcrystallinecellulose 410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesiumstearate 5.0 500.0 mg/capsule (iii) Capsule ‘Compound X’ 10.0 Colloidalsilicon dioxide 1.5 Lactose 465.5 Pregelatinized starch 120.0 Magnesiumstearate 3.0 600.0 mg/mL (iv) Injection 1 (1 mg/mL) ‘Compound X’ (freeacid form) 1.0 Dibasic sodium phosphate 12.0 Monobasic sodium phosphate0.7 Sodium chloride 4.5 1.0N Sodium hydroxide solution q.s. (pHadjustment to 7.0-7.5) Water for injection q.s. ad 1 mL (v) Injection 2(10 mg/mL) ‘Compound X’ (free acid form) 10.0 Monobasic sodium phosphate0.3 Dibasic sodium phosphate 1.1 Polyethylene glycol 400 200.0 0.1NSodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water forinjection q.s. ad 1 mL mg/can (vi) Aerosol ‘Compound X’ 20 Oleic acid 10Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000Dichlorotetrafluoroethane 5,000 wt. % (vii) Topical Gel 1 ‘Compound X’5% Carbomer 934 1.25%   Triethanolamine q.s. (pH adjustment to 5-7)Methyl paraben 0.2%  Purified water q.s. to 100 g (viii)Topical Gel 2‘Compound X’ 5% Methylcellulose 2% Methyl paraben 0.2%  Propyl paraben0.02%   Purified water q.s. to 100 g (ix)Topical Ointment ‘Compound X’5% Propylene glycol 1% Anhydrous ointment base 40%  Polysorbate 80 2%Methyl paraben 0.2%  Purified water q.s. to 100 g (x) Topical Cream 1‘Compound X’ 5% White bees wax 10%  Liquid paraffin 30%  Benzyl alcohol5% Purified water q.s. to 100 g (xi) Topical Cream 2 ‘Compound X’ 5%Stearic acid 10%  Glyceryl monostearate 3% Polyoxyethylene stearyl ether3% Sorbitol 5% Isopropyl palmitate 2% Methyl Paraben 0.2%  Purifiedwater q.s. to 100 g

These formulations may be prepared by conventional procedures well knownin the pharmaceutical art. It will be appreciated that the abovepharmaceutical compositions may be varied according to well-knownpharmaceutical techniques to accommodate differing amounts and types ofactive ingredient ‘Compound X’. Aerosol formulation (vi) may be used inconjunction with a standard, metered dose aerosol dispenser. Thecompounds described herein may also be delivered in combination withdelivery systems such as nanoparticles, micelles, or liposomes.Additionally, the specific ingredients and proportions are forillustrative purposes. Ingredients may be exchanged for suitableequivalents and proportions may be varied, according to the desiredproperties of the dosage form of interest.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments, exemplary embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims. The specific embodiments provided herein are examplesof useful embodiments of the present invention and it will be apparentto one skilled in the art that the present invention may be carried outusing a large number of variations of the devices, device components,methods steps set forth in the present description. As will berecognized by one of skill in the art, methods and devices useful forthe present methods can include a large number of optional compositionand processing elements and steps.

Although the present invention has been described with reference tocertain embodiments thereof, other embodiments are possible withoutdeparting from the present invention. Although the description hereincontains a plurality of specificities, these should not be construed aslimiting the scope of the invention but as merely providingillustrations of some of the presently preferred embodiments of theinvention. The spirit and scope of the appended claims should not belimited, therefore, to the description of any specific embodimentscontained herein. All embodiments that come within the meaning of theclaims, either literally or by equivalence are intended to be embracedtherein. Furthermore, the advantages described above are not necessarilythe only advantages of the invention, and it is not necessarily expectedthat all of the described advantages will be achieved with everyembodiment of the invention.

All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference, to theextent each reference is at least partially not inconsistent with thedisclosure in this application (for example, a reference that ispartially inconsistent is incorporated by reference except for thepartially inconsistent portion of the reference). References citedherein are incorporated by reference to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when compounds areclaimed generically, it should be understood that compounds known andavailable in the art prior to Applicant's invention, including compoundsfor which an enabling disclosure is provided in the references citedherein, are not intended to be included in the compounds claims herein.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples of the disclosure and should not be takenas limiting the scope of the invention. Rather, the scope of thedisclosure is defined by the following claims. We therefore claim as ourinvention all that comes within the scope and spirit of these claims.

What is claimed is:
 1. A pharmaceutical composition comprising: (i) adeoxynyboquinone (DNQ) compound having the formula:

wherein R₁, R₂, R₃, and R₄ are each independently —H or —X—R; each X isindependently a direct bond or a bridging group, wherein the bridginggroup is O—, —S—, —NH—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—, or alinker of the formula —W-A-W—, wherein each W is independentlyN(R′)C(═O)—, —C(═O)N(R′)—, —OC(═O)—, —C(═O)O—, —O—, —S—, —S(O)—,—S(O)₂—, —N(R′)—, —C(═O)—, —(CH₂)_(n)— wherein n is 1-10, or a directbond, wherein each R′ is independently H, (C₁-C₆)alkyl, or a nitrogenprotecting group; and each A is independently (C₁-C₂₀)alkyl,(C₂-C₁₈)alkenyl, (C₂-C₁₈)alkynyl, (C₃-C₈)cycloalkyl, (C₈-C₁₀)aryl,(OCH₂—CH₂)_(n)—, wherein n is 1 to 20, C(O)NH(CH₂)_(n)— wherein n is 1to 6, OP(O)(OH)O—, —OP(O)(OH)O(CH₂)_(n)— wherein n is 1 to 6, or(C₁-C₂₀)alkyl, (C₂-C₁₆)alkenyl, (C₂-C₁₆)alkynyl, or —(OCH₂—CH₂)_(n)—interrupted between two carbons, or between a carbon and an oxygen, witha cycloalkyl, heterocycle, or aryl group; each R is independently alkyl,alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl,heterocycloalkyl, heterocycloalkenyl, (cycloalkyl)alkyl,(heterocycloalkyl)alkyl, (cycloalkyl)heteroalkyl,(heterocycloalkyl)heteroalkyl, aryl, heteroaryl, (aryl)alkyl,(heteroaryl)alkyl, hydrogen, hydroxy, hydroxyalkyl, alkoxy,(alkoxy)alkyl, alkenyloxy, alkynyloxy, (cycloalkyl)alkoxy,heterocycloalkyloxy, amino, alkylamino, aminoalkyl, acylamino,arylamino, sulfonylamino, sulfinylamino, COR^(x), —COOR^(x), —CONHR^(x),—NHCOR^(x), —NHCOOR^(x), —NHCONHR^(x), —N₃, —CN, —NC, —NCO, —NO₂, —SH,-halo, alkoxycarbonyl, alkylaminocarbonyl, sulfonate, sulfonic acid,alkylsulfonyl, alkylsulfinyl, arylsulfonyl, arylsulfinyl, aminosulfonyl,R^(x)S(O)R^(y)—, R^(x)S(O)₂R^(y)—, R^(x)C(O)N(R^(x))R^(y)—,R^(x)SO₂N(R^(x))R^(y)—, R^(x)N(R^(x))C(O)R^(y)—, R^(x)N(R^(x))SO₂R^(y)—,R^(x)N(R^(x))C(O)N(R^(x))R^(y)—, carboxaldehyde, acyl, acyloxy, —OPO₃H₂,—OPO₃Z₂ where Z is an inorganic cation, or saccharide; wherein eachR^(x) is independently H, OH, alkyl or aryl, and each R^(y) isindependently a group W; wherein any alkyl or aryl contains optionallysubstitution of one or more hydroxy, amino, cyano, nitro, or halogroups; or a salt or solvate thereof; (ii) a poly-ADP ribose polymerase(PARP1) inhibitor; and (iii) a pharmaceutically acceptable diluent,carrier, or excipient, wherein said composition comprises an effectiveamount of each of the DNQ compound and the PARP1 inhibitor that yields asynergistic cytotoxic effect against cancer cells.
 2. The pharmaceuticalcomposition of claim 1, wherein the PARP1 inhibitor is selected from thegroup consisting of AG-014699 (Rucaparib), ABT-888 (Veliparib), BSI-201(Iniparib), AZD2281 (Olaparib), AG14361 and INO-1001.
 3. Thepharmaceutical composition of claim 1, wherein the diluent, excipient,or carrier is water, hydroxypropyl-β-cyclodextrin (HPβCD) or acombination thereof.
 4. The pharmaceutical composition of claim 1,wherein R₁, R₂, and R₃ are each methyl, R₄ is not H or methyl.
 5. Thepharmaceutical composition of claim 1, wherein R₁, R₃, and R₄ are eachmethyl, the group —X—R of R₂ is not —CH₂—Oac, or when R₁, R₃, and R₄ areeach methyl, the R group of R₂ is not acyloxy.
 6. The pharmaceuticalcomposition of claim 1, wherein R₁, R₂, R₃ and R₄ are not each H at thesame occurrence; or R₁, R₂, R₃ and R₄ are not each alkyl at the sameoccurrence, wherein the alkyl is methyl.
 7. The pharmaceuticalcomposition of claim 1, wherein R₁, R₂, R₃, and R₄ are each a(C₁₋₂₀)alkyl group, a (C₂₋₂₀)alkyl group, a (C₃₋₂₀)alkyl group, a(C₄₋₂₀)alkyl group, a (C₅₋₂₀)alkyl group, or a (C₁₀₋₂₀)alkyl group. 8.The pharmaceutical composition of claim 1, wherein the DNQ compound hasthe structure:

or a salt or solvate thereof.
 9. A pharmaceutical composition comprisinga DNQ compound having the structure:

or a salt or solvate thereof, and a PARP inhibitor, wherein saidcomposition comprises an effective amount of each of the DNQ compoundand the PARP1 inhibitor that yields a synergistic cytotoxic effectagainst cancer cells.
 10. The pharmaceutical composition of claim 9,wherein the PARP inhibitor is selected from the group consisting ofAG-014699 (Rucaparib), ABT-888 (Veliparib), BSI-201 (Iniparib), AZD2281(Olaparib), AG14361 and INO-1001.